501 PartIII Spr2012

Biochemistry 501 Spring 2012 Part 3 Course Packet
Biochemistry 501 Part III: Biosynthesis and metabolic regulation Study Guide How to approach this section of the course: Personalize it! Essentially everything that we will discuss occurs in your own body. So, as you go about your day eating, sleeping, exercising, going through stress, or relaxing, you can imagine how all of these events play out in your organs and individual cells. For every pathway, try to imagine how it is regulated under various physiological conditions. Many of you are headed towards careers in health-related fields (e.g. medicine, veterinary medicine, dietetics, etc.). Your patients and clients are avid readers of self-help books. Nutrition books are among the largest best sellers in the book world. So, you will be asked questions about claims made in these books. If you have any of these books, it is interesting to re-read them in light of your new knowledge. Read before lectures. Read slowly and re-read. The reason to read more than once is that the first time through, youll be learning many new words. It is like learning a language; you must have a vocabulary and an understanding of the key components of a pathway before you can understand how pathways are regulated and interrelated or how they connect to physiology. The terms will be used in lecture; you do not want to be lost for lack of vocabulary. The lectures will repeatedly refer to the names of enzymes. But, on the exam, the name of the enzyme will be accompanied by the reaction it catalyzes (e.g. glucokinase: Glucose G-6-P), so you need not systematically memorize all the enzyme names. Readings: The textbook (Lehninger by Nelson and Cox 5th edition) is quite large and therefore contains far more information than can be covered in any one course. You need to read in detail the topics covered in lecture. The lecture will not cover every detail because the textbook is quite well-written. Instead, the lectures are aimed to guide your learning and help you to integrate and contextualize the information. You need to learn the primary information through your reading. The lectures do not always follow the exact order of presentation of the textbook chapters. So, you may have to jump around between sections of the textbook to prepare for a particular lecture topic. The regulation of metabolism is mentioned in chapter 23, but also throughout various sections of the book. If you have a favorite biochemistry textbook other than Lehninger that covers all of these topics, you may use that book. In addition to the readings, I would like to suggest that you pay particular attention to the notes in the big handout that I gave you. The major points of each lecture are provided in the preamble for each lecture. Within the lecture itself you will find short statements in note form that describe reactions of pathways or enzyme cascades or other biological effects. Read your notes carefully. If you have trouble come and see me. Be able to recognize and explain the chemical transformations that occur in the pathways. You do not need to know each individual reaction, but given a reaction you should be able to understand what is happening.
Listed below are several process you will learn. You should be able to associate them with the appropriate plant or animal tissues and the cellular compartments where they occur. Pentose phosphate pathway Fatty acid synthesis Triacylglycerol synthesis Ketone body formation Cholesterol synthesis Chylomicron formation Chylomicron transport Triacylglycerol transport VLDL formation HDL formation LDL formation Cholesterol transport Gluconeogenesis Glycogen synthesis Glycogenolysis Insulin secretion Glucagon secretion Epinephrine secretion Amino acid synthesis Nucleotide biosynthesis
When studying bear in mind that catabolism of one metabolite drives synthesis of another type of metabolite. Forward and reverse pathways of metabolism are not identical, i.e. glycolysis and gluconeogenesis; fatty acid oxidation and fatty acid synthesis. Pathways do not occur as isolated events. They do not occur in a vacuum, i.e. fatty acid oxidation is mandatory for gluconeogenesis. The energy required to drive gluconeogenesis comes from fatty acid oxidation. Understand the regulated steps of: Fatty acid synthesis Gluconeogenesis Glycogen synthesis Glycogen breakdown Know the physiological purposes of these processes, i.e. what is the physiological purpose of gluconeogenesis? Understand the concepts of feedback or product inhibition, feed forward activation, the committed step concept and covalent modification, i.e. phosphorylation and dephosphorylation. Do not forget the biological effects of lipids and how these effects are brought about. Exam: The exam for this section of the course will be matching , labeling, true/false and multiple-choice. It will emphasis regulatory pathways and integration of metabolism. Although you will not have to draw any chemical structures, you may have to recognize a structure and select its name. Good luck Cholesterol synthesis Amino acid biosynthesis Nucleotide biosynthesis
Professor Ntambis Textbook References Part III: Biosynthesis and metabolic regulation
Class 1
Lecture Topic Pentose phosphate pathway
Fifth Edition Pgs 558-563
Fourth Edition Pgs 549-555
Synthesis of fatty acids and phospholipids
Pgs 805-817; 820-831
Pgs 787-798; 808-815
Synthesis of ketones, sterols and isoprenoids
Pgs 666-668, 831-836
Pgs 650-652; 815-820
Cholesterol regulation, lipoproteins
Pgs 836-845
Pgs 820-827
Biological activities of lipids
Pgs 357-369; 817-820; 905, 908, 1144
Pgs 357-363; 800-803; 885; 888-889; 1109
Regulation of blood glucose I: Gluconeogenesis
Pgs 551-558
Pgs 543-549
Regulation of blood glucose II: Glycogen metabolism
Pgs 594-602
Pgs 560-570
Regulation of blood glucose III: Hormonal control
Pgs 602-608
Pgs 575-591
Biosynthesis of amino acids and porphyrins
Pgs 860-878; 909
Pgs 840-862; 889
10
Nucleotide biosynthesis
Pgs 885-896
Pgs 862-878
11
Integration of metabolism
Parts I, II and III
Parts I, II, and III
3 Options for Glucose-6-P

Glucose!
Glucose-! 6-phosphate!
Pentose Phosphate Pathway

(Ch 14. pgs 558 563)
Glucose-! 1-phosphate!
Fructose-! 6-phosphate!
6-Phospho-! gluconate!
Glycogen Pyruvate Glycogen! Glycolysis!
Ribose! 5-phosphate Pentose Shunt!
Roles of Pentose Phosphate Pathway
Overview of Pentose Phosphate Pathway
Net reaction:
Generates NADPH for biosynthesis Anabolic! Generates pentose-P for nucleotides Catabolic! Degrades pentoses from diet Catabolic!
3 Glucose-6-P + 6 NADP+ + 3 H2O ! 6 NADPH + 6 H+ + 3CO2 + 2 Fructose-6-P + Glyceraldehyde-3-P
Operates in two phases:

Oxidative: NADPH and Ribulose-5-P Non-oxidative rearrangements : Ribose-5-P, and Fructose-6-P and Glyceraldehyde-3-P
p1
NADPH
Nicotinamide Adenine Dinucleotide Phosphate
Pentose Phosphate Pathway

Oxidative
Intermediates
Products
Oxidative Reactions
Reaction 1: Glucose-6-phosphate dehydrogenase
HO H HO H H C C C C C CH2O P H OH H OH O
NADP+ NADPH, H+
Oxidative Reactions
Reaction 2: 6-phosphoglucono-lactonase
C H HO H C C C C
O OH H OH O H HO H H
C C C C C
O OH H OH O
H2O
COOH+
H HO H H
C C C C
OH H OH OH
Glucose-6-phosphate dehydrogenase
CH2 O P
CH2O P
CH2O P
Glucose-6-phosphate!
6-phosphoglucono-lactone!
6-phosphoglucono-lactone!
6-phosphogluconate!
p2
Oxidative Reactions
Reaction 3: Phosphogluconate dehydrogenase
COO! !
Overview of Pentose Phosphate Pathway
Net reaction:
+ NADP! ! + NADPH, H! !
H! HO! H! H!
C! C! C! C!
OH! H! OH! OH! CO2! ! ! H! H!
C! C! C!
CH2OH! !! O! OH! OH!
3 Glucose-6-P + 6 NADP+ + 3 H2O ! 6 NADPH + 6 H+ + 3CO2 + 2 Fructose-6-P + Glyceraldehyde-3-P
Operates in two phases:

Oxidative: NADPH and Ribulose-5-P Non-oxidative rearrangements : Ribose-5-P, and Fructose-6-P and Glyceraldehyde-3-P
CH! O P! 2!
CH! O P! 2!
6-phosphogluconate!
Ribulose-5-phosphate!
Total for oxidative reactions: two NADPH per glucose
Non-oxidative Reactions
H H C C
H+
Carbon skeleton rearrangements in the Pentose Phosphate Pathway
OH O OH OH
H+
Phosphopentose epimerase!
H
H+
!!
H H
C C
!!
H C C H H C C
H C C C OH OOH OH
C H2OPO32 Ribulose-5phosphate (Ru5P)
OH
Phosphopentose isomerase!
H+
!!
H
OOH OH
Substrate!
!!
H C H H H C C C OH OH OH O
H H C C HO H C C OH O H OH
C5!+!C5! ! ! ! ! C7 +!C3! ! ! ! ! C5 +!C4! ! ! ! ! Sum:! 3 C5! ! !
C3 +!C7! ! ! ! ! C6 +!C4! ! ! ! ! C6 +!C3! ! ! ! ! 2C6 +!1C3! ! ! ! ! !
Transketolase! Transaldolase! Transketolase!
C H2 OPO32 2,3-Enediolate intermediate
C H2OPO32 1,2-Enediolate intermediate
C H2OPO32 Xylulose-5phosphate (Xu5P)!
C H2OPO32 Ribose-5phosphate (R5P)!
Product!
Product!
p3
First Transketolase Reaction

Transfered group:!
Transaldolase Reaction
H! C 2!
Transfered group:!
H! C 2! C!
OH! O!
OH! O! H!
CH2OH
C! HO! C!
Production of Sedoheptulose-7-phosphate and Glyceraldehyde-3-phosphate

O C H H H C C C CH2 H OH OH OH O P
! OH! ! C! O! HO! C! H! + H! C! OH! CH! O P! 2 !

CH2
HO
Transketolase TPP
H
O C C CH2 H OH O P
! H! H! H!
! OH! ! ! O! C! H! C! OH! C! OH! C! OH! CH! O P! 2 !

CH2 C
Xylulose5-phosphate
Ribose5-phosphate
Glyceraldehyde3-phosphate
Sedoheptulose7-phosphate
!! ! ! O! HO! C! H! H! C! OH! H! C! OH! +! H! C! OH! CH! O P! 2 ! Sedoheptulose-! 7-phosphate!

CH2 OH C
!! ! ! O! O C H HO! C! H! Transaldolase! H C OH C H +! H! C! OH! H C OH H C OH H! C! OH! CH2 O P CH2 O P CH! O P! ! 2 ! Erythose-4-! Glyceraldehyde-! Fructose-6-! phosphate (E4P)! 3-phosphate! phosphate (F6P)!
O C
Second Transketolase Reaction

H! C 2! C! OH! O!
Flux of Metabolites through PPP

Flux of metabolites through Pentose Phosphate Pathway (PPP) depends on metabolic needs
Ribose-5-phosphate
CH2 OH O H OH OH O P
Transfered group:!
CH2 C HO H C C CH2 O H
OH
O C H OH OH O P
NADPH production
Generation of Fructose-6-phosphate and Glyceraldehyde-3-phosphate Recycled through shunt
HO
C C C CH2
OH O P
+! !
H H
C C CH2
Transketolase TPP
H
C C CH2
H OH O P
+! ! !
H H
Energy generation
Generation of Fructose-6-phosphate and Glyceraldehyde-3-phosphate Glycolysis and TCA cycle
Xylulose5-phosphate
Erythose4-phosphate
Glyceraldehyde3-phosphate
Fructose6-phosphate
p4
Modes of Pentose Phosphate Pathway

Mode 2: NADPH synthesis
Mode 1: Nucleotide synthesis

Gluconeogenesis
Xu5P
G6P
G6P!
6PG!
Ru5P!
R5P!
Nucleotides!
NADPH! NADPH + CO2!
6PG
Ru5P
R5P
S7P
G3P
! !
E4P
X5P
! !
G6P
G3P
DHAP
F6P
FBP

Mode 3: Energy generation
Regulation of Pentose Phosphate Pathway
Oxidative Phase
Non-oxidative Phase
Glycolysis
!
Pyruvate
TCA cycle CO2
G3P
G6P
!
CO2
Ru5P
!
F6P
!
G3P
NADPH,
ATP!
! !
GTP NADH FADH2!
Rate-limiting step: G-6-P dehydrogenase Rate is controlled by substrate availability: NADP+
CO2
Ac-S-CoA
CO2
p5
Reactive Oxygen Species
Reactive Oxygen Species and Human Disease
O2 + e-
O2H2O2
superoxide peroxide
" Reaction " Reaction
O2 + 2e- + 2H+ O2 + 3e- + 3H+ O2 + 4e- + 4H+
with DNA causes mutations with proteins denatures them are a major source of ROS lack DNA repair
H2O + OH hydroxyl free radical 2 H 2O
" Mitochondria " Mitochondria " Mitos
very susceptible to mutation
Defenses Against Reactive Oxygen Species (ROS)
Role of NADPH and Glutathione as Anti-oxidants
Superoxide dismutase:
2 O2 + 2H+ 2H2O2 2GSH + H2O2 H2O2 + O2 2H2O + O2 GSSG + 2H2O
POB, 5th ed., Page 559
Peroxidase, Catalase: Glutathione peroxidase:
GSH refers to reduced glutathione, a tripeptide of the amino acids: glycine, glutamate and cystine
p6
Inherited Glucose 6-P Dehydrogenase Deficiency in Erythrocytes

Under normal circumstances, no symptoms Confers resistance to malaria parasite Plasmodium falciparum Basis of resistance: oxidative stress to parasite Antimalarial drugs lead to hemolytic anemia by causing oxidative stress (primaquine) Ditto for sulfa drugs (antibiotics)
Fava Beans, Falafel, Favism, and Divicine

Genetic predisposition to anemia caused by divicine in fava beans Individuals have defect in glucose 6-phosphate dehydrogenase Divicine produces oxidative stress, leading to! Inability to repair oxidative damage to red cells, leading to! Hemolytic anemia (potentially fatal)
Wernicke-Korsakoff Syndrome
Severe hemolytic anemia Uncoordinated movement, eye movement, amnesia Common in alcoholics Ethanol inhibits thiamine uptake Some individuals with W-K Syndrome have Gluc 6-P dehydrogenase with high Km for thiamine Thiamine deficiency inhibits pentose phosphate pathway, reducing NADPH
Reactive Oxygen Species in Phagocytosis
After phagocyte engulfs bacterium, etc. Dramatic increase in O2 uptake (respiratory burst) Production of reactive oxygen: NADPH + 2O2 NADP+ + 2O2 + H+
(Powerful microbicide)
p7
Synthesis of Fatty Acids (and Sterols)
- Acetate (Acetyl-CoA) is precursor
Synthesis of Fatty Acids and Phospholipids
- Repetitive condensations - Endergonic - ATP - Reductive - NADPH
Intracellular Localization of Fat Synthesis
Malonyl-CoA Synthesis
- Rate-limiting step in fatty acid synthesis

- Citrate stimulates - Palmitoyl-CoA inhibits
p8
Acyl Carrier Protein (ACP)
Fatty Acid Synthase Complex
MW! 10,000 Da Acetyl-CoA + ACPSH"Acetyl-S-ACP + CoASH Malonyl-CoA+ACPSH"Malonyl-S-ACP+CoASH 2 separate trans-acylases
- Intermediates remain attached to enzyme - 7 enzyme activities
Condensation of Acetyl- and Malonyl-
Reduction of Acetoacetyl-S-ACP
p9
Dehydration
Second Reduction
Second Round of Condensation
Second Round of Condensation
p10
Fatty Acyl- Chain Elongation
Elongation and Desaturation of Fatty Acids

The elongation and desaturation of fatty acids are carried out by accessory enzyme systems
desaturation (in plants only)
Palmitate 16:0!
elongation
Stearate 18:0
desaturation
Oleate 18:1(#9)!
Linoleate 18:2(#9,12)
desaturation
elongation
Palmitoleate 16:1(#9)
Longer saturated fatty acids!
desaturation (in plants only)
desaturation
$-Linolenate 18:3(#6,9,12) %-Linolenate 18:3(#9,12,15)

elongation
Other polyunsaturated fatty acids!
Eicosatrienoate 20:3(#8,11,14)
desaturation
Arachidonate 20:4(#5,8,11,14)!
FA Synthase: Multi-enzyme Complex
Source of NADPH
p11
Acetate export from mitochondria
Regulation of FA synthesis at the first step
See POB Fig. 21-10
Coordinate Regulation of FA Metabolism
Fatty Acid Metabolism

Synthesis Cytoplasm ACP-SH NADPH D-3-Hydroxybutyryl-CoA 3C + 2C " 4C + 1C (malonyl-ACP) Regulators: Citrate Palmitate Breakdown Mitochondria CoASH NAD+, FAD L-3-Hydroxybutyryl-CoA 4C " 2C + 2C (acetyl-CoA) Regulators: Insulin
p12
Biosynthesis of Triglycerides and Phospholipids

Glycerol 3-phosphate dehydrogenase
CH2 OH | C=O | 2CH2 OPO3
Acyltransferase
Acyltransferase
CH2 O C R1 O | R 2 C O CH O | CH2 OPO23 H2O
Phosphatidate phosphatase
!! Glycerol 3-phosphate! !!! ! !! ! ! ! !!! ! ! ! !! !! !! ! ! ! !!! !! !! !! Monoacylglycerol 3-phosphate! !! ! ! ! ! !!! ! ! ! !! !! !! ! ! ! ! !!! !! !! !! Phosphatidate! !! ! !! !!! ! !!! !!!! ! !!! ! ! !!
NAD + CH2 OH | HO CH | 2CH2 OPO3 CoA-S C R 1 O CoA-SH CH2 O C R1 | HO CH O | CH2 OPO23 CoA-S C R 2 O CoA-SH P1
! ! !
! !! ! ! ! ! ! !! !! !
Dihydroxyacetone phosphate
NADH + H +
!!
Synthesis of TG, PC, PE from Phosphatidate

! CoA-S ! ! !! !C !R !
O
3
P!! ! ! CH!!!O !C !!R!! 1,2-Diacylglycerol! CDP-Ethanolamine! |! R!! ! ! ! C ! O ! CH ! O! ! |! Acyltransferase! Phosphoethanolamine! CH!! ! ! OH ! CMP! CoA-SH! transferase! O! O! CDP-choline! Phosphocholine! CH!!! ! !! ! O !C R ! CH!! ! ! !! ! O !C R ! ! O! O! transferase! |! |! CMP! R!! ! !! ! C O ! CH ! O! ! R!! ! ! ! C ! O ! CH ! O! ! |! |! O! CH!! ! ! !! ! ! O !C R CH!! ! ! ! ! !! !! !! !! ! O ! P|!! O ! CH CH ! N H |! CH!! ! ! !! ! O !C R ! O! O! O-! |! R!! ! ! ! C ! O ! CH ! O! ! Triacylglycerol! |! CH!!! ! !! ! !! !! !! !! ! O ! P O ! CH ! CH ! N (CH ) |! O-! Phosphatidylcholine!
1
Phosphatidate phosphatase O
! !
H2O
!! !
1
33
! NH!! ! CH! ! ! ! ! !R!! OC! N! O! |! R!! ! ! ! ! ! C O CH ! O! ! O! O! N! |! CH! ! !O ! ! !O ! ! ! ! ! ! ! ! P ! P O CH ! ! ! O! | |! O!! O!! H! H! H! H! N!! OH! OH! CDP-diacylglycerol! |! HO ! ! ! ! ! ! CH ! CH COO!! Inositol! Serine! Phosphatidylinositol synthase! Phosphatidylserine synthase! [Prokaryotes! [ E. coli ]! CMP! CMP! and Eukaryotes]! O! O! CH!! ! ! ! ! ! !! OC R ! O! |! CH! ! ! ! ! ! ! !! OC R ! O! R!! !C ! ! !CH !O ! O! ! |! |! H! OH! ! R!! !C ! ! !CH !O ! O! ! N!H! ! CH!!! ! ! ! ! ! O P O ! H! | | |! OH! H! CH!! ! ! ! ! ! ! ! ! ! ! ! O P O CH ! CH COO!! ! O!! OH!HO! |! OH! -! H! O! H! H! Phosphatidylserine! Phosphatidylinositol!
PPi O
2 2 1 2 2 2 + 2 2 1 2 1 2 2 + 3 2 2 2
CTP: phosphatidate cytidylyltransferase
! !
CTP
p13
Biosynthesis of Sphingolipids
! Palmitoyl-CoA! C ! !!! !! ! !! ! (CH ) CH CoA-S! Serine!

O
2 14 3
CoA-SH, CO!! !!! !! ! !! C! |! H!N!! ! ! ! C!! H ! & -Ketosphinganine | OH ! CH!! ! ! NADPH + H! !

2
(CH 2 ) 14 CH3
HO 3 CH (CH 2 ) 14 CH3 | Sphinganine H3N+ 2C H | CH2 OH
NADP!! ! !! !! !!! ! ! !! ! !! !!!!!!!!! ! !!!! !

+
! !
Fatty acyl-CoA
CoA-SH
O HO CH (CH 2 ) 14 CH3 | Ceramide, R C NH C H | containing CH2 OH Sphinganine
! ! ! !! !!! ! ! !! ! ! !! !! !! !! ! ! ! !!!! ! !
O HO CH (CH2 )14 CH 3 | Ceramide, R C NH C H | containing CH 2 OH mixed-function oxidase (animals)
! ! ! !! !!! ! ! ! ! ! ! ! ! ! ! !! !! ! ! ! !! ! ! Sphinganine! ! ! !
UDP-Glc
Gangliosides GM1!
&1,4
6% of nervous system lipids!
&1,4 Gal! Glu! ceramide!
Gal-NA!
UDP
Activated sugars
Gal! &1,3! Nan!
%2,3
O HO CH CH = CH (CH 2) 12 CH 3 | Ceramide, R C NH C H containing | CH 2 OH Sphinganine Phosphatidylcholine head group attachment
! ! ! !! ! ! ! ! ! !! !! ! !! ! ! ! !
! ! !!! ! ! !! ! ! ! ! !
O HO CH CH = CH (CH 2) 12 CH 3 | Cerebroside R C NH C H | CH2 O Glc
! ! ! !! ! ! !!! ! ! !! ! ! ! ! ! ! !! ! ! ! ! !! ! !! !
6% of nervous system lipids!
Activated sugars! O! HO! CH !CH = CH (CH2! 12 !!CH3! ! ! ! ! ! )! ! |! Cerebroside! R !C !NH !C !H ! ! ! ! ! |! CH2!!O !Glc! ! !
Diacyglycerol
O HO CH CH = CH (CH2)12 CH3 | R C NH C H O Sphingomyelin | CH 2 O P O CH2 CH2 N(CH3)3
! ! ! !! ! ! !!! ! ! !! ! ! ! ! ! !! !! ! ! ! ! !! ! !! !! !! !! ! !! ! !!!! O!
p14
Genetic Defects in Ganglioside Metabolism
Tay-Sachs, Fabrys, Sandhoffs Diseases of Spingolipid Catabolism
Gauchers, Niemann-Pick Diseases
p15
Ketogenesis
The final product from the breakdown of fatty acids is acetylCoA, which as we saw in the previous sections enters the TCA cycle. It is important to learn at this point that this acetyl-CoA will enter the TCA cycle only if fat and carbohydrate metabolism are appropriately balanced. However, if fat breakdown predominates, the acetyl-CoA in the LIVER AND ONLY IN THE LIVER undergoes a different fate. This is because entry of acetyl-CoA into the TCA cycle depends on the availability of oxaloacetate leading to the formation of citrate. In fasting or in diabetes, oxaloacetate is used to synthesize glucose and is thus not available to form citrate with acetyl-CoA. Under these conditions the acetyl-CoA is diverted to the formation of acetoacetate, 3-hydoxybutyrate and acetone referred to as KETONE BODIES.
Synthesis of Ketones, Sterols and Isoprenoids
Physiological Conditions that Promote Ketogenesis

Fasting - starvation Diabetes Ketogenic diet High fat Newborn
Ketones or ketone bodies Acetoacetate ! "-hydroxybutyrate Acetone!
Acetoacetate and "-hydroxybutyrate are specialized circulating fuels produced by the liver to supply certain tissues (e.g., brain, nerve, muscle) with non-glucose derived fuel especially during fasting.
p16
Fatty Acyl-CoA
"-oxidation
mitochondrial matrix
Use of Ketone Bodies
Produced in the liver Used by heart muscle and kidney Starvation: brain Excess Ac-CoA - low OAA in TCA Diabetics: acetone breath
p17
Synthesis of Cholesterol and Isoprenoids Cholesterogenesis

Cholesterol, a steroid component of eukaryotic membranes and a precursor of steroid hormones, is also formed from acetyl-CoA. The major sites for cholesterol synthesis are the liver, intestine, adrenal cortex and the gonads. Unlike ketone body synthesis, synthesis of cholesterol takes place in the cytoplasm. !
Nobel Prize 1964
All Carbons in Cholesterol Come from Acetate
Origin of Cytoplasmic Acetyl-CoA Precursor

Cytoplasm
!
CoA, OAA ATP
Mitochondria
! !
! ! !
Cholesterol
Citrate OAA + Acetyl-CoA
Citrate Acetyl-CoA Citrate lyase
!
Sphingolipids
+!ACC! !
Malonyl-CoA
! !
Phospholipids
Pyruvate
FAS
Palmitate
!
Triglycerides
p18
Tissue Distribution of Cholesterogenesis

Major sites: Liver Small intestine Adrenal cortex Gonads! Minor sites: Other tissues!
Stages 1 and 2 of Cholesterol Synthesis
~ all cell types can synthesize cholesterol, BUT derive it from plasma lipoprotein secreted by the liver and intestine. If the liver and intestine do not provide an adequate supply, DE NOVO synthesis of cholesterol is activated in other cells.!
POB fig. 21-33
Stages 3 and 4 of Cholesterol Synthesis
Stage 1: Acetyl-CoA to Acetoacetyl-CoA
POB fig. 21-33
POB fig. 21-34
p19
Stage 1: Acetoacetyl-CoA to HMG-CoA

!
Stage 1: HMG-CoA to Mevalonate
Site of Statin Action
POB fig. 21-34
POB fig. 21-34
Stage 2: Mevalonate to Activated Isoprenes
POB fig. 21-35
POB fig. 21-35
p20
POB fig. 21-35
POB fig. 21-35
Stage 3: Isoprenes (5-C) to Farnesyl (15-C)
Stage 3: Two Farnesyl-PP (15-C) Condense Head-to-Head to Squalene (30-C)
POB fig. 21-36
POB fig. 21-36
p21
Stage 4: Squalene Cyclization Forms Steroid Nucleus
Stage 4: Squalene Cyclization Forms Steroid Nucleus
POB fig. 21-37
Final Touches Convert Lanosterol to Cholesterol
Synthesis of Cholesterol Esters
POB fig. 21-38
p22
Regulation of HMG-CoA Reductase Synthesis by Cholesterol
Regulation of Cholesterol Synthesis
SREBP: Sterol response element binding protein SCAP: SREBP-cleavage activating protein!
HMG-CoA reductase, etc.

POB fig. 21-43 POB fig. 21-44
Other Isoprenoids
Cholesterol As Steroid Precursor

Cholesterol (27C) Vitamin D hormone Bile salts Pregnenolone (21C)
Progestagens (21C) Glucocorticoids gluconeogenesis inflammation Mineralocorticoids renal absorption of Na+, Cl-, HCO3Sex hormones poetry wars
p23
Protein Prenylation
p24
Cholesterol vs. Coronary Heart Disease
Cholesterol regulation, lipoproteins
Cholesterol Regulation, Lipoproteins

Cholesterol and other lipids are transported in blood to specific targets by a series of lipoproteins. Exogenously derived cholesterol and triglycerides are transported in the blood to tissues by lipoprotein particles called chylomycrons. Endogenously synthesized triglycerides and cholesterol are packaged and transported from the liver to adipose tissue by very low density lipoproteins (VLDL). After delivering its contents to the tissues the VLDL is converted first to intermediate density lipoprotein (IDL) and then into low density lipoprotein (LDL) which is taken up by the liver and peripheral tissue cells by receptor mediated endocytosis.
Cholesterol Regulation, Lipoproteins

The LDL receptor is a transmembrane protein spanning the plasma membrane of cells, binds LDL and mediates its entry into cells. Absence of LDL receptor causes hypercholesterolemia (i.e., leads to marked elevation in the level of plasma LDL cholesterol, deposition of cholesterol on blood vessel walls, and heart attacks). Excess cholesterol from tissues is carried back to the liver by high density lipoprotein (HDL). This cholesterol can be excreted to the outside via bile acids, hence reducing the level of cholesterol from the body. So HDL carries the good cholesterol, whereas LDL carries the bad cholesterol.
p25
fasting!
fed!
Lipoprotein Particles in Postprandial Plasma
Characteristics of the Major Classes of Lipoproteins in Human Plasma!

Lipoprotein Class
Chylomicrons and remnants
Major Lipids
Dietary triacylglycerols
Apoproteins
A-I, A-II, B-48, C-I C-II, C-III, E
Density (gcm-3)
<0.95
Particle Diameter ()
800-5000
VLDL
Endogenous triacylglycerols, cholesteryl esters, cholesterol Cholesteryl esters, cholesterol, triacylglycerols Cholesteryl esters, cholesterol, triacyl-glycerols Cholesteryl esters, cholesterol
B-100, C-I, C-II, C-III, E B-100, C-III, E
0.95-1.006
300-800
IDL
1.006-1.019
250-350
LDL
B-100
1.019-1.063
180-280
HDL
A-I, A-II, C-I, C-II, C-III, D, E
1.063-1.210!
50-120
Postprandial - after a meal
p26
Plasma Lipoproteins
Plasma Lipoprotein
Phospholipid monolayer ApoB-100
Triacylglycerols
Cholesterol esters
Free cholesterol (unesterified)
Amphipathic & Non-polar Lipids
LDL Receptor
The LDL receptor is a transmembrane receptor with five different functional domains!
LDL binding domain cytoplasm
p27
A Model for Plasma Triglyceride and Cholesterol Transport in Humans
VLDL - IDL - LDL Pathway
LDL Receptors Control Plasma, LDL Production and Uptake

Normal:

Familial Hypercholesterolemia:
p28
Familial Hypercholesterolemia
Plasma cholesterol: >800 mg/dl in homozygotes >300 mg/dl in heterozygotes Frequency: 1/500 people

High Cholesterol Diet:
tendon xanthomas
eyelid xanthomas
Atherosclerosis plaque buildup in a blood vessel
Atherosclerosis plaque buildup in a blood vessel
1. 2. 3. 4.
Infection damages inner lining of artery (red) As part of the healing process cholesterol is deposited on the damaged area (yellow) A thin layer of congealed blood forms over the cholesterol (red) This layer prevents the body from removing the cholesterol plaque
1. 2. 3. 4.
Infection damages inner lining of artery (red) As part of the healing process cholesterol is deposited on the damaged area (yellow) A thin layer of congealed blood forms over the cholesterol (red) This layer prevents the body from removing the cholesterol plaque
p29
Consequences
Strategy for Treatment

Reduce hepatic cholesterol level Feed Lovastatin or Compactin to inhibit HMG-CoA reductase Feed Cholestyramine to reduce hepatic cholesterol pool Cholesterol Bile salts Bile salts bind to Cholestyramine and thus are not reabsorbed and returned to the liver
HO O
OR
Normal section cut of an artery Tear in artery wall
!
O
!
O
Fatty material is deposited in vessel wall
Lumen becomes blocked by a blood clot
!
Lovastatin!
H in compactin
H3C
!! !
Lowered hepatic cholesterol level induces receptor synthesis leading to increased LDL receptor level
Statins Decrease LDL Levels
Lipitor (Atorvastatin)
LDL Receptor
LDL Clearance LDL Levels
p30
Biological Activities of Lipids

The objective of this lecture is to develop an understanding of the general principles of signal transduction pathways involving lipids. Emphasis will be on: 1. The phosphoinositide cascade leading to the generation of intracellular second messengers diacylglycerol (DAG) and inositol triphosphate (IP3). DAG is an endogenous activator of protein kinase C, and IP3 causes a release of Ca2+ (3rd messenger) from intracellular stores. 2. Steroid hormones: These hormones produce most of their effects by inducing the transcription of tissue-specific genes. 3. Eicosanoids: This is the term for 20 carbon unsaturated fatty acids derived from arachidonic acid which make up a class of hormone-like biomodulator substances. Arachidonic acid itself is derived from linoleic acid, an essential fatty acid in our diet.
Biological activities of lipids
Types of Lipids
Storage Lipids: Membrane Lipids: Neutral fat: Triacylglycerols Polar lipids: Phospholipids Glycolipids Sphingolipids Sterols Steroids Prostaglandins A, D, and K Bile acids: Waxes Pigments Scents and more!! For digestion
PIP2!
DG!
Phosphatidylinositol
Receptor-triggered hydrolysis of PIP2
Hormones: Fat soluble Vitamins:!
PLC
!
IP3
IP3
p31
Hormone Receptor
Phosphoinositide cascade
Phospholipase C Plasma membrane
Phosphoinositide cascade
A wide variety of biological effects are mediated by the Phosphoinositide cascade Glycogenlysis in liver cells Histamine secretion by mast cells Serotonin release by blood platelets Smooth muscle contraction Visual transduction in invertebrate photoreceptors Insulin secretion by pancraetic islet cells
ER
Protein kinase C
Many others!
Steroid Hormones
Cholesterol Progesterone: Androgens: Estrogens: Testosterone Glucorcorticoids: Estradiol Aldosterone (mineralocorticoid)
Regulates re-absorption of Na+, Cl-, HCO3- in the kidney Male and female sex hormones. Influence secondary sexual characteristics. Regulate female reproductive cycle.
Steroid Hormones
Prepares uteral lining for embryo RU 486 has similar structure Testosterone development of male sexual characteristics Development of female sex characteristics Tamoxifen has similar structure Promote gluconeogenesis and glycogen formation Act on distal kidney tubule to promote re-absorption of Na+
Pregnenolone
Progesterone Cortisol (glucocorticoid)

Affects protein and carbohydrate metabolism Suppresses immune response, inflammation, and allergic responses.
Corticosterone (mineralocorticoid)
Mineralocorticoids: (aldosterone)
!
Vitamin D3: Involved in Ca++ metabolism Lack of Vitamin D causes rickets in children and osteomalicia in adults
p32
Steroid Hormone Receptors

All steroid hormone receptors share substantial sequence homology and functional domain substructure!
Receptor Y Zinc Zinc Hormone!
Model for Steroid Receptor Action
H! N! ! Y! K! R! ! G 2 0! ! D! R! ! V! DNA I! K! ! W! S! 50 ! ! !T ! S! C! C! C C! 10 ! ! Q! Q! 0! !! 6 V! E! N!! Zn! T! ! !!! Zn! A! A! G! A! ! P! C! C! C! C! 30! 40! 70! 80! RLRKCYEVGMMKGGIRKDRRGG! MKETRY! KAFFKRSIQGHNDYM! G S A Y D N H3N+
Receptor!
Hormone! response! element!
!! ! ! !
Transcription DNA binding activation (highly conserved) (variable length)
COO-
!!
Hormone binding (variable length)
Specificity of Binding to Steroid Receptors in the Design of Drugs

Progesterone vs RU 486
RU 486 has similar structure as progesterone It is used as an antagonist of progesterone in early termination of pregnancy !
Vitamin D
Vitamin D is a derivative of cholesterol and a precursor to a hormone essential in calcium and phosphate metabolism in vertebrates.
Progesterone
RU 486 Mifepristone
Estrogen vs Tamoxifen
Tamoxifen has similar structure as estrogen It competes with estrogen in binding to the estrogen receptor, but the tamoxifen receptor complex is inactive in gene regulation. The cancer cells that depend on estrogen for growth will be killed off. So tamoxifen is used in the treatment of breast cancer in humans.
Vitamin D3 is formed in the skin in a photochemical reaction driven by UV light. It is also abundant in fish oils and is added to commercial milk as a nutritional supplement. The active form of the vitamin is 1,25-dihydroxycholecalciferol. Deficiency of vitamin D leads to defective bone formation, resulting in the disease rickets.!
Estrogen
Tamoxifen
p33
CH3 H3C H3C CH CH2 CH2 CH2
H C CH3 CH3
Eicosanoids
! ! ! !
Phospholipid containing arachidonate
! ! !! ! !!
Phospholipase A2
!
5
Lysophospholipid
HO
7-Dehydroxycholesterol
UV
!
H CH2 CH2 CH2
25
20
14
11
COO-
! !! =!
!
2O2
CH3 H3C H2 C
1
! Prostaglandin! endoperoxide! synthase!

11 14
! COO 20!
Arachidonate, 20:4(!5, 8, 11, 14 )
!!
COO-
aspirin, ibuprofen
CH
C CH3
CH3
HO
Vitamin D 3 Cholecalciferol
!!
PGG2
!
OH
OOH
Liver enzyme oxidizes C-25
!
CH3 H3C CH CH2 CH2 CH2
25
Prostaglandin endoperoxide synthase

O
CH3 C CH3
Kidney enzyme oxidizes C-1!
! ! !
COO-
HO
H2 C
1
O OH
PGH2
1,25-Dihydroxycholecalciferol
HO
!
Other Prostaglandins
Thromboxanes
Eicosanoids
COOO Ser OH C CH3
Ser O C CH3
Linear Pathway from Arachidonate to Leukotrienes

COOO OH
14 20 11 8 5 1
COO-
+
Salicylate!
HOO
Arachidonate!
lipoxygenase
Prostaglandin endoperoxide synthase
Asprin (Acetylsalicylate)!
Acetylated, inactivated enzyme

H3C
O2
!!
12
COO-
O2 lipoxygenase
!!
OOH
5
!! !
CH
CH3
!!
12-Hydroperoxyeicosatetraenoate (12-HPETE)
!
COO-
! !!
Other leukotrienes
CH2
5-Hydroperoxyeicosatetraenoate (5-HPETE)
CH H3C
!
COO-
Leukotriene A4 (LTA4)
!! !
!!
LTC4 LTD4
Ibuprofen!
p34
Functions of Eicosanoids
The inflamatory response Regulation of pain and fever Regulation of blood pressure Induction of blood clotting Control of reproductive functions Regulation of the sleep-wake cycle
Most eicosanoids are extremely short lived. They have metabolic half lives of seconds to minutes. This means that the eicosanoids target neighboring cells not distant organs.
p35
Gluconeogenesis
Regulation of blood glucose I: Gluconeogenesis
The regulation of carbohydrate metabolism is necessary to maintain a constant level of blood glucose. There are essential links to lipid and amino acid metabolism.
Blood glucose levels must be controlled within very tight limits. The process of glucose homeostasis is related to the fact that the brain requires glucose. A level of glucose below about 1.5 mM results in coma due to lack of ATP production. By contrast, hyperglycemia leads to hyperosmolar, hyperglycemic coma. This condition is observed in some older diabetic people. For an understanding of caloric homeostasis, it is important to remember the close interrelations between carbohydrate, fatty acid and amino acid metabolism.
The formation of glucose from non hexose precursors is called gluconeogenesis (formation of new sugar). Gluconeogenesis is a universal pathway found in all animals, plants, and microorganisms. The reactions are the same in every case. The important precursors in animals are lactate, pyruvate, glycerol and most of the amino acids. In higher animals gluconeogenesis occurs mainly in the liver, and to a lesser extent in the kidney cortex. Gluconeogenesis converts fats and proteins to glucose in germinating seeds, so unlike animals, plants can convert acetyl-CoA derived from fatty acid oxidation via the Glyoxylate cycle.
p36
Fatty acid is not a substrate for net glucose production

Certain tissues have a continual requirement for glucose: Brain and nerve Red blood cells Kidney medulla Testis
Fatty Acids !-oxidation Acetyl-CoA C2 OAA no net carbon flux TCA Cycle Citrate
Therefore under conditions of carbohydrate deprivation, glucose is synthesized from non-carbohydrate sources (process = gluconeogenesis).
CO2 CO2
Gluconeogenesis
Pyruvate is a substrate for glucose production

Fatty Acids !-oxidation Pyruvate Glucose Acetyl-CoA C2 C4 OAA TCA Cycle Citrate C4
Gluconeogenesis is an energy requiring process The energy required to fuel gluconeogenesis comes from "-oxidation of fatty acids The amino acids provide the carbon skeletons for glucose synthesis
CO2 CO2
p37
Protein
amino acids
glucose
Glycerol as a Glucogenic Substrate

Adipose
Liver Protein
Glyceraldehyde-3-P
Amino acids Protein degradation & transamination
F-1-6-P2
Gluconeogenesis
Glycolysis
p38
Reactions of Gluconeogenesis
Not simply a reversal of glycolysis
Highly negative #G of 3 reactions Overall: #G = -82 kJ / mol (in the cell) Independently regulate synthesis and breakdown
Three bypasses of irreversible steps

Four separate enzymes Energy input Other 7 steps are glycolysis reverse! Forward Reverse
Glycolysis!
Pyr. kinase P-fructokinase Hexokinase
#Go
31 14 17
#Go
+ 31 + 14 + 17
Gluconeogenesis!
Pyr. carboxylase PEP carboxykinase F-1,6-bis-Pase G6Pase
#Go
+ 0.8 16.0 12.0
Bypass One
Bypass one: Pyruvate $ PEP (2 steps) Overall: Pyruvate + 2 ATP $ PEP + 2ADP +Pi
Compartmentalization
Pyruvate carboxylase in mitochondria, PEP carboxykinase in cytosol Pyruvate transported directly
O-
Pyruvate Carboxylase
Biotin cofactor
O C CH3 O C O-O ATP ADP + Pi O C CH2 O C O C
Pyruvate
HCO3- + ATP
Oxaloacetate
Malate shuttle: OOA transport
PEP Carboxykinase
GTP-driven decarboxylation No net CO2 fixation
O -O C CH2 O C O C OGTP CH2 GDP + CO 2 PO32O C O C O-
Oxaloacetate
Phosphoenolpyruvate: PEP
p39
Bypass Two and Three!

Bypass Two: Fructose-1,6-bis-P $ Fructose-6-P
Fructose-1,6-bis-phosphatase: hydrolysis Important regulatory step
Regulation of Gluconeogenesis / Glycolysis

Two conflicting pathways:
Reciprocal regulation avoids futile cycle High energy $ stimulates gluconeogenesis Low energy $ stimulates glycolysis!
Bypass Three: Glucose-6-P $ Glucose

Glucose-6-phosphatase: hydrolysis
Net reaction:
2 Pyruvate + 4 ATP + 2 GTP + 2 NADH + 6 H2O $ Glucose + 4 ADP + 2 GDP + 6 Pi + 2 NAD+ + 2 H+
Major control point:
F-6-P % F-1,6-bis-P
Committed step (glycolysis) ATP / AMP ratio controls glycolysis / gluconeogenesis But varying ATP / AMP insufficient to explain regulation & another regulator
Energy cost:
Reverse of glycolysis: 2 ~ P, 2 NADH Cost gluconeogenesis: 6 ~ P, 2 NADH Extra cost: 4~P
Regulation of Gluconeogenesis / Glycolysis

Most important regulator: Fructose-2,6-bis-P (1980)
Regulation by Fructose-2,6-bis-P
Not an intermediate: regulator only F-2,6-bP allosterically: Activates PFK1 (glycolysis) Inactivates FBPase1 (gluconeogenesis) Tandem enzyme of opposite reactions: PFK 2 makes F-2,6-bis-P FBPase 2 breaks down F-2,6-bis-P F-6-P affects opposing reactions High F-6-P (high glucose): feed-forward stimulation of glycolysis Phosphorylation also controls which activity used (hormonal) ATP
PFK-1
Gluconeogenesis
Fructose 6-phosphate F26BP Fructose 1,6-bisphosphate
Pi
FBPase-1
ADP
H 2O
Glycolysis
p40
The Cori Cycle

Production of lactate in muscle - vigorous activity:
Aerobic: Glycolysis and TCA Anaerobic: Glycolysis $ Lactate
Liver takes over metabolic burden:

Takes lactate from muscle, returns glucose
p41
Regulation of Blood Glucose: Glycogen Metabolism

In a wide range of organisms, excess glucose is converted into polymeric forms for storage and transport. The principal storage forms of glucose are glycogen in animals and microorganisms and starch in plants. In animals glucose itself is transported in the blood, but the transport form in plants is sucrose. The substrate for glycogen biosynthesis is UDP-glucose
Regulation of blood glucose II: Glycogen metabolism
Regulation of Blood Glucose: Glycogen Metabolism

35 nm
Glycogen synthesis occurs virtually in all animal tissues, but is especially prominent in liver and skeletal muscle. In liver glycogen serves as a reservoir of glucose readily converted into blood glucose for distribution to other tissues, whereas in muscle glycogen is broken down via glycolysis to provide ATP for muscle contractions. Glycogen is stored in the cytoplasm in glycogen granules (10 to 40 nM). Glycogen granules contain several glycogen molecules and the enzymes for glycogen synthesis and degradation.!
Branched chains of glucose residue in glycogen
Catalytic face
Monolayer of specific enzyme molecules covering glycogen molecule
Regulatory face Glycogen phosphorylase dimer
p42
Glycogen
Glycogen is a branched polymer in which most of the backbone glucose residues are linked in !-1,4-glycosidic bonds. The branch points are created by !-1,6-glycosidic bonds. !1 " 6 branch point
Branched form of Glycogen

Reducing end Non-reducing ends
HOCH2 H HO H OH H OH OH H O
HOCH2 H H OH H OH H OH O
HOCH2 H H OH H O H H OH CH2 O
! -1,6
Non-reducing
!
H H OH H
! -1,4
HOCH2 O H H OH O H H OH H O H H OH O HOCH2 H H OH H OH H OH O HOCH2 H H OH H OH H OH O H
HOCH2
HOCH2 H OH H OH H OH O H H OH H O H H OH R
HO
Non-reducing ends
Different Enzymes for Synthesis and Breakdown

Degradation - phosphorolysis
[Glucose]n+1 + Pi " [Glucose]n + G-1-P
Glycogen Breakdown
Glycogen breakdown involves three enzymatic activities: Glycogen phosphorylase Glycosyl transferase !-1,6-glucosidase
Non-reducing end
6 CH OH 2 5
!
CH2OH CH2OH H O H OH H OH H OH H OH H OH H OH
H
4
H OH H
3
OH H 1 OH
2
H O
HO
Glycogen chain (glucose)n
!!
Pi
Synthesis
[Glucose]n + UDP-glucose " [Glucose]n+1 + UDP
H HO H OH CH2OH H OH OH H O OP O O-
Glycogen phosphorylase
Non-reducing end
CH2OH H OH H OH O
!
CH2OH H OH H OH H OH
+!
H OH H
HO
Glucose-1-phosphate
Glycogen shortened by one residue (glucose)n1
! ! !!
p43
Non-reducing ends!
(!1" 6) linkage
Glucose-6-Phosphatase*
! ! ! !
In liver and kidney Not found in muscle or adipose tissue Essential for glucose secretion Defines distinct roles for glycogen in liver vs. muscle:
Glycogen
! !
Glycogen phosphorylase
Glucose-1-phosphate molecules!
Transferase activity of Debranching enzyme
Glucose!
(!1" 6) Glycosidase activity of Debranching enzyme
Glycogen " G-1-P " G-6-P " glycolysis
Glycogen " G-1-P " G-6-P !
Muscle ATP
Unbranched (!1" 6) polymer; Substrate for futher phosphorylase action
*! Pi Blood glucose
Glycogen Breakdown Liver vs. Muscle
Glycogen Synthesis
Four stages of glycogen synthesis:
Conversion of glucose-6-phosphate to glucose-1-phosphate by phosphoglucomutase Glucose phosphate is converted to UDP-glucose by UDP-glucose pyrophosphorylase UDP-glucose is the immediate donor of glucose residues in the reaction catalyzed by glycogen synthase Creation of branches by transfer of segments to 1,6, linkages by glycosyl-4:6 transferase
p44
Glycogen Synthesis (activation of sugar)

HOCH2 H HO H H OH O H H O OH O P OO-
Glycogen Synthesis (activation of sugar)
Glucose 1-phosphate
+!
O -O P OO O P OO O P OO Uridine
HOCH2 H HO H H OH O H H O OH O P OO O P OO Uridine
PP PPii
UDP-glucose!
Glycogen Synthase Reaction

(1 " 4)-terminal chains of glycogen
Branching enzyme
!(1" 6) Increases solubility and rate of degration!
p45
Tyr194
! !
Plant Starch
Glycogenin! UDP-glucose! UDP!

glycogen synthase
Protein-tyrosineglucosyltransferase (glycogenin) !
UDP-glucose! UDP!
Amylose: Amylopectin:
Not branched !(1" 4) Branched like glycogen, but less frequent branching
glycogenin
Glycogen! synthase!
glycogen synthase glycogen branching enzyme
UDP-glucose! UDP!
glycogenin
UDP-glucose! Mg2+! ! UDP!
Glycogen particle!
Branched form of Glycogen

!1 " 6 branch point Reducing end Non-reducing ends
Non-reducing ends Glycogen Phophorylase A
p46
Regulation of Blood Glucose

Normal level :! 80-120 mg 100 ml! = ~ 1 g/l ! 5 mM !
Regulation of blood glucose III: Hormonal control
Humans have about 5 liters of blood so Total glucose in blood is about 5 g! Basal metabolism : 50-100 kcal / hour! Since glucose " 4 kcal / gm, total blood glucose would support basal metabolism < 1 hour
Glucose Tolerance Test

15!
Endocrine Pancreas; pancreatic islet
10! Blood! glucose! (mM)! 5!
Diabetic!
Normal!
1! 1! 2! 3! Hour(s)! 4!
p47
Glucose Regulation of Insulin Secretion
Fluctuations in Insulin
Rate-Limiting Steps in Glucose Clearance

Muscle & Adipose Liver

Muscle & Adipose Liver
Glucose
Glucose
Glucose
Glut4
Glut2
Glucose
Glucose
Glucose glucokinase Glucose-6-P
Glucose
Glucose glucokinase Glucose-6-P
Glut4 Transport Phosphorylation

Low Km Acutely regulated by insulin
Glut2
High Km Not regulated by insulin
p48

Muscle & Adipose
Glucose transport
Glucokinase
A Glucose Buffer
+Insulin
increased Vmax
Glut4
+!
Insulin (fast)
Blood Glucose
Glucose concentration
glucokinase
Glucose
Glucose-6-P
glucose-6-phosphatase
Insulin ! "glucokinase = more glucose buffering Diabetes or starvation ! #glucokinase= less glucose buffering
Summary of Insulin Action
Ought you give glucose to a starving person?
Glucose transporters move from cytosol to the membrane and allow glucose to enter cell
p49
Regulation of Glycogen Metabolism: The Action of Glucagon and Epinephrine

Both Hormones :
Produced in response to low [ glucose ] in blood Bind to cell-surface receptors of target cells Increase intracellular cAMP " increase in blood glucose
cyclic AMP-- A universal starvation signal
Organ-specific control :!
Glucagon Source : Primary Target :! Pancreas Liver! Epinephrine Adrenal gland Muscle!
cyclic AMP-- A universal starvation signal
Activation of ser / thr Protein Kinase by cAMP

Cyclic AMP!
Adenylate cyclase
Inactive Protein Kinase A!
+!
Regulatory Catalytic subunit subunit Complex of cyclic AMP and Active catalytic regulatory subunits! subunit
Further amplification : 1 cAMP " many P-proteins Kinase phosphorylates different targets : coordinates response to hormone
p50
Amplification of Hormonal Signal
Regulation of Glycogen Metabolism
Hormonal Regulation of Glycolytic and Gluconeogenic Enzymes

Both pathways regulated at key control step : F-6-P ! F-1,6-bis-P Key regulator of this step : F-2,6-bis-P Tandem enzyme that interconverts : F-6-P ! F-2,6-bis-P is under phosphorylation control
Regulation of Liver Glycolysis
p51
PKA Regulation of PFK-2/F-2,6P2ase
Summary of Blood Glucose Regulation

Decrease in Blood Glucose! Increase in Blood Glucose!
Release of insulin Release of glucagon
Glucagon binds to membrane receptor
! !
Insulin binds to membrane receptor
! ! ! !
Activation of adenylate cyclase Increase in cAMP Activation of cAMP-dependent kinase and inhibition of glycogen synthase
Increased activation of glycogen synthase
! ! ! !
Exocytosis of glucose permease molecules
! ! ! !
Increased permeability to glucose
Activation of glycogen phosphorylase
Removal of glucose from blood! and its storage as glycogen!
Release of glucose into blood!
p52
Biosynthesis of Amino Acids and Porphyrins

All amino acids are derived from intermediates in glycolysis, the citric acid cycle or the pentose phosphate pathway. Nitrogen enters these pathways by way of glutamine and glutamate.
Biosynthesis of Amino Acids and Porphyrins
Some pathways are simple, others are not. Ten of the amino acids can be synthesized using one or a few enzymatic steps. Others such as aromatic amino acids use more complex reactions. Whereas plants and bacteria can synthesize all the twenty amino acids, mammals can synthesize only ten of the simple ones, the so-called nonessential amino acids. The remainder, the essential amino acids, must be obtained from the diet.
Transamination: General Reaction NH3! ! R1! ! C! COO! H! R1! !

PLP Aminotransferase (! transaminase)
Role of "-KG in Transamination

Alanine Pyruvate
COO! O! C!
O! C! COO!
H3N! !!
COO! CH! CH3! ! +! COO! O! C! CH2! ! CH2! ! COO!
+!
O! R2! ! C! COO!
+!
NH3! ! C! COO! H!
PLP
Alanine !! aminotransferase H3N! CH!
CH3! ! +! COO!
R2! !
CH2! ! CH2! !
PLP = Pyridoxal phosphate, from vitamin B6
"-ketoglutarate
Glutamate
COO!
p53
Non-Essential Amino Acid Pathways
Non-Essential Amino Acid Pathways
1. PLP-dependent transamination
Oxaloacetate "-Ketoglutarate Pyruvate Aspartate Glutamate Alanine
3. Phenylalanine hydroxylase (tetrahydrobiopterin cofactor)

Phenylalanine Tyrosine
2. Amidation of Glu, Asp

Glutamate Aspartate Glutamine Asparagine
4. Proline by reversal of Pro degradation

Glu Glu-semialdehyde Schiff base to close ring
3-Phosphoglycerate Family (Ser, Gly, Cys)
3-Phosphoglycerate Family (Ser, Gly, Cys)
Fig. 22-12!
p54
Arginine Synthesis
Branched Path to Tyr, Phe, Trp

PEP + Erythrose 4-Phosphate
(4 steps)
Ultimately, from "-ketoglutarate Via urea cycle In human children, arginine is required in diet; adults make enough via urea cycle
Shikimic acid
(3 steps)
Chorismic acid! Prephenate

Glutamine! PRPP !
Glutamate
Serine!
Tyrosine
Phenylalanine
Tryptophan
Summary : Origins of Amino Acids

All 20 amino acids derive from 6 central intermediates :!
Ribose-5-P gly
Essential Amino Acids in Humans

Val Met His Leu Phe Lys Ile Thr Trp Arg
!
ser
Glucose!
! !
his
! ! !
Erythrose-4-P
3-PGA! PEP! Pyruvate! "-KG

Shikimate
cys
!
ala val
! !
trp
Chorismate Prephenate
! !
Adults (9)
Children (10)
asn
!
asp
leu
! met!
lys thr
phe
OAA!
gln
glu
! !
pro
tyr
arg
!
Mnemonic : Very Many Hairy Little Pigs Live In The Torrid Argentina
ile
p55
Porphyrin
OOC! H2C! !! H2C! !! H3C! !! C! C! C! H! C! C! C! C! COO! CH2! ! CH2! ! CH3! !
C! N! HC! C! N! H2C! !! C! C! C! H! CH! 3! C! C! H!
N! C! Fe! N! C! C! C! C! H! CH2! ! C! CH3! ! CH!
Heme Fe-protoporphyrin!
Synthesis of Porphyrins
Heme b Chlorophyll
First Step of Porphyrin Biosynthesis
hemoglobin, catalase, cytochromes
p56
ALA Synthesis in Plants
Next: Condensation of ALA
Porphyrias
Genetic Defects in Heme Synthesis Congenital erythropoietic porphyria
deficiencies in protoporphyrin synthesis and chelatase
Acute intermittent porphyria

affects liver deficiencies in porphobilinogen deaminase build up of ALA and porphobilinogen
p57
Glutathione: A Unique Tripeptide
GSH Is a Redox Buffer
Reduced GSH !
Oxidized GSSH #-Glu-Cys-Gly S !

NADPH !
X
Synthetase not synthase!!!!
! #-Glu-Cys-Gly S !
S ! #-Glu-Cys-Gly
Amino Acid Metabolism (cont.)

COO
GABA Synthesis
Inhibitory neurotransmitter
COO H3N+ C CH2 C CH NH H
Nicotinic acid
HO C N H CH CH2CH2+NH3
COO H3 N+! C! H! CH2! CH2!

C CH2COO
H! CO2 H3N! C! H! CH2! CH2! COO!
Serotonin
PLP
COO!
Tryptophan
N H
CH
Glutamate
#-aminobutyric acid (GABA)
Indole acetic acid (auxin)
p58
Histamine Synthesis
HO CH2
H 3N + C H
Catecholamines
OH COO HO HO CH2 CH2 NH CH3
Tyrosine!
Epinephrine !
Methyltransferase SAM
OH
CO2
PLP-dependent decarboxylation
O2 Hydroxylase H 2O
H 3N + HO HO CH2 C H COO
SAhomocysteine
HO HO
CH2
CH2
NH2
DOPA!
Norepinephrine !
H 2O O2 Hydroxylase
Histidine !
Histamine !
CO2
Decarboxylase
HO HO CH2 CH2 NH2
The synthesis and action of histamine are major drug targets
Dopamine!
Nitric Oxide Synthesis

Vasodilators (e.g. acetylcholine) stimulate synthesis of nitric oxide (NO) NO is formed by the enzyme, NO synthase
Rxn :
Arginine
NOS
Redox cofactors
Citrulline + NO
Nitroglycerin is metabolized to NO
p59
Nucleotide Biosynthesis
Nucleotides play a variety of important roles in all cells. First, they are precursors of DNA and RNA. Second, ATP and to a certain extent GTP are essential carriers of chemical energy. Third, nucleotides are components of the cofactors NAD, FAD, S-adenosylmethionine, and coenzyme A, as well as activated biosynthetic intermediates such as UDPglucose and CDP-diacylglycerol. Some, such as cAMP and cGMP, are also second messengers.
Use in medicine: Anticancer agents: methotrexate, fluorouracil Anti-AIDS drug: azidothymidine (AZT)
There are two types of pathways leading to nucleotides: 1. The de novo pathways: Here nucleotide biosynthesis begins with metabolic precursors: amino acids, ribose-5-phosphate, CO2 and NH3. 2. Salvage pathways: these pathways recycle the free bases and nucleosides released from nucleic acid metabolism.
Review of Nucleotides
Definitions: Nucleoside: nitrogenous base + sugar Nucleotide: nitrogen base + sugar + phosphate! Base formula !
NH2 N
Purines
Names and abbreviations of nucleic acid bases, nucleosides, and nucleotides ! Base X-H ! Nucleoside X - ribose ! Nucleotide X - ribose phosphate !
!
N
Adenine Ade A!
Adenosine Ado A
Adenylic acid Adenosine monophosphate AMP!
Ribonucleoside!
Ribonucleotide!
N X
!
Guanine Gua G! Guanosine Guo G Guanylic acid Guanosine monophosphate GMP!
O N N Deoxyribonucleoside Deoxyribonucleotide!
!
N
Sugar moiety: ribose (RNA) vs. deoxyribose (DNA) Nitrogenous bases: Purines and pyrimidines!
H 2N
N X
p60
Pyrimidines
Names and abbreviations of nucleic acid bases, nucleosides, and nucleotides ! Base formula !
NH2! N O! N X! O! H! N N X!
Purine Nucleotide Biosynthesis

Nucleotide X - ribose phosphate !
Base X-H !
Nucleoside X - ribose !
Origin of atoms:
CO2! glycine! C N
1! 2! 6! 5! C 4! 7! 9!
Cytosine Cyt C!
Cytidine Cyd C
Cytidylic acid Cytosine Monophosphate CMP!
Aspartate amine!
N
8! C
O!
Uracil Ura U!
Uridine Urd U
Uridylic acid (only RNA) Uridine monophosphate UMP!
formate!
C formate!
3!
N H
O
H! N N dX! CH3!
O!
Thymine Thy T!
Deoxythymidine dThd dT
Deoxythymidylic acid (only DNA) Deoxythymidine monophosphate dTMP!
Glutamine amide!
Ribose-5-P
Inosine Monophosphate (IMP)
Built up from ribose directly Pathway of reaction: Ribose-5-P Inosine monophosphate (IMP)
!-D-Ribose-5-phosphate
Glycine + ATP ribose phosphate pyrophosphokinase activation ATP 1 AMP GAR synthetase 3 ADP +Pi
5-Phosphoribosyl-!-pyrophosphate (PRPP)
amidophosphoribosyl transferase committed for purine synthesis Glutamine + H2O 2 Glutamate + PPi
Glycinamide ribotide (GAR)

N10-Formyl-TH GAR transformylase 4 THF
"-5-Phosphoribosylamine
Ribose-5-phosphate
p61
Ribose-5-P

Formylglycinamide ribotide (FGAR)
Ribose-5-P

5-Aminoimidazole ribotide (AIR) CO2
7
AIR carboxylase
Enzyme 3, 4 & 6 are with same peptide to keep all intermediates around
ATP + Glutamine + H2O FGAM synthetase

5
ADP + Glutamate + Pi
Carboxyaminoimidazole ribotide (CAIR)
ATP + Aspartate
Formylglycinamidine ribotide (FGAM)
AIR synthetase 8
ADP + Pi 5-Aminoimidazole-4-(N-succinylocarboxamide) ribotide (SACAIR)
ATP AIR synthetase

6
ADP + Pi + H2O 5-Aminoimidazole ribotide (AIR)
adenylosuccinate lyase 9
Fumarate
Ribose-5-P

5-Aminoimidazole-4-carboxamide ribotide (AICAR)
N1 0-Formyl-THF
AICAR transformylase 10
OOC

Generation of AMP and GMP from IMP
O Aspartate + GTP GDP + Pi N CH2 CH NH2 N N N N Ribose-5-phosphate! COO H N N NAD+ + H2O N Ribose-5-phosphate! NADH + H2O
THF
Adenylosuccinate synthetase!
IMP !
IMP dehydrogenase!
O H N N H N N Ribose-5-phosphate!
5-Formaminoimidazole-4-carboxamide ribotide (FAICAR)
Adenylosuccinate
IMP cyclohydrolase 11
Xanthosine monophosphate (XMP)

Glutamine + ATP +H2O!
H2O
fumarate!
Adenylosuccinate lyase!
Glutamate + AMP +PPi!
GMP synthase!
Hypoxanthine precursor to adenine and guanine

Inosine monophosphate (IMP)
NH2 N N N N Ribose-5-phosphate! H N
O N N N Ribose-5-phosphate!
H 2N
AMP
GMP
p62
Origin of Pyrimidine Atoms

O H 2N C O
Carbamoyl phosphate
Pyrimidine Biosynthesis
O P O O
Aspartate
Pi Aspartate transcarbamoylase
H 2O NAD+ NADH + H+ O HN CH CO2
Glutamine Amide
Aspartate
C ! N
HCO3 !
3 4 5C
O
CTP
ATP
Orotate
O
C2
6C
N H
UMP
O
HN HCO3
OMP
H 2O N
PPi
PRPP
Ribose 5-phosphate
Interconversion of Nucleotides
BASE
Interconversion of Nucleotides Adenylate kinase (myokinase)

P
H 2C O H H H OH
P +
P +
AMP + ATP
ADP + ADP
H HO
Nucleoside monophosphate kinase e.g. UMP + ATP UDP + ADP Diphospho-Nucleoside kinases
UDP + ATP ADP + GTP
UTP + ADP ATP + GDP
p63
Reduction of Ribonucleotides
Free Radicals in Biochemistry
CH2
4
BASE O H
3 1
CH2
4
BASE O H
3 1
Homolytic cleavage of CH bond
H
2
H
2
H HO OH
H HO H
.! C . !
C + H
NDP
Substrates: UDP, CDP, ADP, GDP Free Radical Mechanism!
dNDP
seen where C-H cleavage occurs at unactivated carbons
Ribonucleotide reductase
Examples of Free Radical Biochemical rxns

!
Flavins and coenzyme Q in e- transfers B12 catalyzed reactions adenosylcobalamin Oxygenation of alkanes Cytochrome P450!
Regulation of Ribonucleotide Reductase Activity

Levels of dNTPs in the cell must be tightly controlled and balanced
Deficiencies are lethal Excesses are mutagenic
C H
C X
C X
C H
Levels of dNTPs maintained by feedback control of ribonucleotide reductase Enzyme has two allosteric sites that control both the enzymes activity and its substrate specificity
p64
Ribonucleotide Reductase
Origin of Thymine Diphospho-Nucleoside kinases dUDP + ATP dUTP + ADP dCDP + GTP dCTP + GDP dCTP deaminase
dCTP dUTP + NH3
and dCTP
Regulated by levels of dTTP

The E.coli enzyme is a tetramer composed of two homodimers Reducing equivalents are donated from NADPH via Thioredoxin and glutathione via Glutaredoxin
dUTPase
dUTP dUMP + PPi
Substrates: UDP, CDP, ADP, GDP
dTMP Synthesis
dUMP Thymidylate synthase
N5,N10-Methylene Tetrahydrofolate
Inhibitors of dTMP Synthesis

dTMP
H 2N HN
Dihydrofolate
H N N CH2 NH CH3
O C NH
COO CH CH2 CH2 COO
NH2
Methotrexate!
O
Dihydrofolate reductase
HN O N
Serine hydroxymethyl transferase
5-uorouracil!
Tetrahydrofolate
p65
Inhibitors of dTMP Synthesis
Nucleotide Degradation and Salvage
Catabolism of purine nucleotides leads to the production of uric acid

Dietary nucleic acids
Free purine bases are salvaged to make nucleotides.
Purine Nucleotide Breakdown
Nucleotide Base Salvage Enzymes
Adenosine phosphoribosyltransferase
Adenine + PRPP # AMP + PPi
Hypoxanthine-guanine phosphoribosyltransferase (HGPRT)

Hypoxanthine + PRPP # IMP + PPi Guanine + PRPP # GMP + PPi
Figure 22-45
p66
Clinical Significance of Purine Metabolism

Severe combined immunodeficiency disease
Deficiency in adenosine deaminase causes 50-fold elevation in dATP levels which inhibits ribonucleotide reductase
Gout is Caused by an Excess of Uric Acid

DNA and RNA in diet
OH OH N N N N H OH
Elion and Hitchings 1988 Nobel Prize Rational design of drugs
N N H N H
Lesch-Nyhan syndrome
Loss of functional HGPRT gene Poor coordination, mental retardation, self-destructive tendencies
Allopurinol
HO
Xanthine Xanthine oxidase

H N N H O
Gout
HN O
Uric acid
N H
p67
Integration of Metabolism
Available Fuels
Sources of Glucose during Fasting
p68
Fuel Levels in Plasma during Fasting
Regulatory Mechanisms
1. Allosteric
changes in affinity of enzyme for substrate very fast local; within cells
2. Phosphorylation
activation/inactivation of enzymes fast global; simultaneous in multiple tissues
3. Induction
changes in number of molecules of enzyme slow global alters enzyme profile of organs
End Product Inhibition

Allosteric regulation : non-covalent binding of regulator to enzyme
Examples : Phosphofructokinase, Aspartate Transcarbamoylase Most common mode :
Problem of Branched Pathway
D A B C G
Linear pathway :
p69
Branched Pathway Regulation

Concerted feedback inhibition :
Inhibition only when both end products are present
Sequential Feedback Inhibition

Branches regulated independently :
D A B C G
F A B C
Cumulative Feedback Inhibition

Glutamate
Covalent Modification
Each end product leads to partial inhibition :
NH3, ATP ADP Glutamine synthetase
Alanine Glycine
Glycogen synthase : Phosphorylation Glutamine synthase : Adenylation Glycosylation
Tryptophan Histidine Glucosamine -6-phosphate
Glutamine
N
AMP CTP Carbamoyl phosphate
p70
Regulation of Enzyme Levels
Subcellular Compartmentation
Cytosol :
Glycolysis Pentose phosphate pathway Fatty acid synthesis
Gene expression Enzyme turnover
Mitochondrial matrix :
Citric acid cycle Oxidative phosphorylation !-Oxidation of fatty acids Ketone body formation
Interplay of both compartments :

Gluconeogenesis Urea synthesis
Organ Specialization
Liver :
Stores glycogen Provides glucose Removes Nitrogen (urea)
Muscle :
Stores glycogen Resting uses fatty acids Active uses glucose - from glycogen
Adipose (fat) cells :

Triacylglycerol storage
Brain :
Glucose is the primary fuel, except during starvation when Ketone Bodies are used
p71
Effects of Insulin
Leptin is a satiety signal
Leptin
satiety signals
Cross-talk between Insulin & Leptin
The Obese Mouse A mutation in the Leptin gene
p72
I WISH YOU ALL GOOD LUCK ON YOUR EXAM
p73
BIOCHEMISTRY 501 PROBLEM SET 1

LECTURES 23-24
1. 2. What are the biological functions of the pentose phosphate pathway? Liver synthesizes fatty acids and lipids (consisting of highly reduced carbon chains) for export to other tissues. Would you expect the pentose phosphate pathway to have low or high activity in this organ? Explain. Would you expect the pentose phosphate pathway to be more or less active in cells that are dividing than in those that are not? Explain. Rat liver is able to metabolize glucose by both the glycolytic and the pentose phosphate pathways. Indicate if the following are properties of glycolytic (G), pentose phosphate (P), both (G+P) or neither (O) pathway a) b) c) d) e) f) 5. 6. NADP is involved H2 is liberated Sedohepulose-7-phosphate is an intermediate Transketolases are involved Thymine pyrophosphate is a cofactor Phosphate esters are intermediates
3. 4.
Explain why we require fats in our diets. Which of the following statements about desaturases in humans are correct? a) b) c) d) They cannot introduce double bonds into a fatty acid that already contains a double bond. They cannot introduce double bonds between the 9 position and the end of the chain. They convert the essential fatty acid linoleate into arachidonate. They use an isozyme of FAD-linked dehydrogenase of the -oxidation cycle to form double bonds.
7.
Acetyl CoA is generated primarily in the mitochondria; however, fatty acid and cholesterol synthesis from acetyl CoA occur in the cytoplasm. Which ONE of the following is currently believed to be the PREDOMINANT mechanism whereby intramitochondrial acetyl-CoA enters the cytosol? a) b) The acetyl group is transferred to carnitine to form acetylcarnitine, which is transported into the cytosol where it reacts with CoA to regenerate acetyl-CoA. Acetyl-CoA reacts with carbon dioxide to form malonyl-CoA, which is transported across the mitochondrial membrane.
p74
c) d) e)
The mitochondrial membrane permits permeation of acetyl-CoA by active transport. Acetoacetyl-CoA formed by the a reversal of the thiolase reaction is deacylated. The acetoacetate thus formed is transported into the cytosol where it is converted back into acetyl-CoA. Acetyl-CoA condenses with oxaloacetate to form citrate, which is transported into the cytosol where it is cleaved to generate acetyl-CoA and oxaloacetate in an ATP dependent reaction.
8.
The fatty acid synthase of mammals is a dimer consisting of identical subunits, each of which contains all the activities necessary to synthesize fatty acids from malonyl CoA and acetyl CoA. Why is a single subunit unable to carry out the reactions?
ANSWERS
1. 2. The pentose phosphate pathway produces pentose phosphates (for nucleic acid biosynthesis) and NADPH (reducing agent for biosynthetic processes). Yes. The pentose phosphate pathway is used by these cells to generate ribose-5phosphate that is used in nucleic acid biosynthesis. Therefore the pathway is active in the dividing cells which require the replication of DNA. The pentose phosphate pathway is very active in liver generating NADPH required for reductive biosynthesis. P; O; P; P; P; G+P Dietary fats provide linoleate and linolenate, which we need for eicosanoid synthesis but we cannot synthesize. b; c. e. Dimerization of the fatty acid synthase allows the enzyme to position all active sites near that of the preceding and succeeding enzyme of the sequence. The flexible pentathenic arm of ACP can reach all of the active sites and carries the growing fatty acid chain from one enzyme active site to the next; the intermediates are not released from the enzyme complex until the finished product is obtained. This channeling of intermediates from one active site to the next increases the efficiency of the overall process.
3. 4. 5. 6, 7. 8.
p75

LECTURES 25-28
1. 2. Why is de novo cholesterol synthesis dependent on the activity of citrate lyase? From the following compounds, identify the intermediates in the synthesis of cholesterol and list them in their proper sequence. a) b) c) d) e) f) g) 3. Geranyl pyrophosphate Squalene Isopentenyl pyrophosphate Mavalonate Cholyl CoA Farnesyl pyrophosphate Lanosterol
Yeast cells growing aerobically are able to synthesize sterols and incorporate them into membranes. However, under anaerobic conditions yeast cells do not survive unless they are provided with an exogenous source of sterols. Explain the metabolic basis of this nutritional requirement. Match the following lipoproteins: Chylomycron VLDL LDL HDL with the appropriate components or properties shown below: a) b) c) d) e) f) g) h) i) Contains apoprotein B-100 Contains apoprotein B-48 Transports endogenous cholesterol esters Transports dietary triacylglycerols Transports endogenous triacylglycerols Is degraded by lipoprotein lipase Is taken up by cells via receptor mediated mechanisms Is a precursor of LDL May remove cholesterol from cells
4.
5. 6.
What effect would adding an inhibitor of glucokinase to liver have on cholesterol synthesis? Why? Which of the following is a lipid with signal-transducing activity?
p76
a) b) c) d) e) 7.
Phosphatidyl choline Phosphatidyl serine Plasminogen activator Phosphatidyl inositol 4,5-bisphosphate Phospholipase A2
Which of the following statements about the phosphoinositide cascade are correct? a) b) c) d) e) The phosphoinositide cascade depends upon hydrolysis of a phospholipid component of the plasma membrane. A polypeptide hormone interacts with the GMI gangalioside on the cell surface to trigger the phosphoinositide cascade. The G-protein system probably acts to transduce the stimulus from the receptor to the phosphoinositidase. Phospholipase C plays a crucial role in the phosphoinositide cascade. The phosphoinositide cascade produces two different messengers.
8.
Which of the following about active Vitamin D is incorrect? a) b) c) d) e) It has the same fused ring system as cholesterol. It requires hydroxylation reactions for its synthesis from cholecalciferol. It is important in the control of calcium and phosphorus metabolism. It can be synthesized from cholesterol in the presence of UV light. It can be derived from diet.
9.
Which of the following statements about eicosanoid hormones are correct? a) b) c) d) e) f) The three major classes of eicosanoid hormones are the prostaglandins, leukotrienes, and thromboxanes. The eicosanoid hormones are derived from arachidonic acid. The eicosanoid hormones are very potent and exert global effects because they are widely distributed by the circulatory system. The prostaglandins have a variety of diverse physiological effects. A prostaglandin precursor is derived from phospholipids. Aspirin inhibits the synthesis of prostaglandin by acetylating prostaglandin synthetase and inhibiting its cyclooxygenase activity.
10.
What is the physiological purpose of gluconeogenesis? What tissues are involved? In the net conversion of pyruvate to glucose how much energy is required in terms of ATP molecules utilized/glucose molecules synthesized? Which of the following statements about gluconeogenesis are correct? a) b) c) It occurs actively in muscle during periods of exercise. It occurs in the liver during periods of exercise or fasting. It occurs actively in adipose tissue during feeding.
11.
p77
d) e) 12.
It occurs actively in kidney during periods of fasting. It occurs actively in brain during periods of fasting.
The following is a sequence of reactions of gluconeogenesis from pyruvate to phosphoenolpyruvate: Pyruvate A Oxaloacetate B Malate C Oxaloacetate D Phosphoenolpyruvate Match the capital letters indicating the reactions of the gluconeogenic pathway with the following statements: a) b) c) d) e) f) g) h) i) j) Occurs in the mitochondria. Occurs in the cytosol. Produces CO2. Consumes CO2. Requires ATP. Requires GTP. Is regulated by acetyl CoA. Requires a biotin cofactor. Is also a reaction of the TCA cycle. Is an anaplerotic reaction
13.
Explain why a deficiency in the enzyme acyl-CoA dehydrogenase would affect gluconeogenesis.
ANSWERS
1. 2. Citrate lyase catalyzes the reaction that releases acetyl-CoA in the cytosol from citrate. Cholesterol is synthesized from acetyl-CoA in the cytosol. Intermediates in order = d-c-a-f-b-g mavalonate-isopentenyl pyrophosphate-geranyl pyrophosphate-farnesyl pyrophosphatesqualene-lanosterol e (cholyl CoA) is not an intermediate, it is a cholesterol derivative precursor of bile salts.
p78
3.
We did not cover this information in lecture. But, for your information: squalene squalene epoxide is catalyzed by a monooxygenase enzyme. The oxygenase adds oxygen to squalene to form an epoxide. Under anaerobic conditions, there is no oxygen, so this step will not occur. Shutting down this step will shut down cholesterol synthesis. Cholesterol is the precursor of steroids, so steroid synthesis will also be prevented. Chylomycron: b, d, f VLDL: a, e, f, h, c, g LDL: a, c, g HDL: c, i, g Glucokinase is the enzyme in liver that catalyzes the production of glucose-6P from glucose (equivalent to hexokinase). There are two reasons why inhibition of glucokinase would affect cholesterol synthesis. 1. 2. glucose-6P is needed for glycolysis to produce acetyl-CoA, the precursor to cholesterol. Glucose-6P is needed for the pentose phosphate pathway for the production of NADPH, the reducing power in cholesterol biosynthesis.
4.
5.
6. 7. 8. 9 10.
d a, c, d, e a a, b, d, e, f Gluconeogenesis exists to produce glucose from non-hexose precursors, mainly for the maintenance of blood glucose levels (5 mM). Gluconeogenesis can occur in several tissues, but only liver and kidney are able to produce glucose for secretion into blood. 6 ATPs (2 are actually GTP) are used to produce one glucose. b, d A) a, d, e, g, h, j B) a, i C) b, i D) b, c, f Acyl-CoA dehydrogenase is an enzyme in fatty acid oxidation (breakdown). -oxidation produces the ATP required to drive gluconeogenesis.
11. 12.
13.
p79

Lectures 29-33
1. 2. 3. 4. A glycogen molecule consisting of 50,000 glucose residues is branched, on average every 10 glucose residues. How many reducing ends does it have? Glycogen is the storage form of glucose in the liver and muscles. Explain why muscle glycogen is not a direct precursor of blood glucose. Explain why the phosphorolytic cleavage of glycogen is more energetically advantageous than its hydrolytic cleavage. A sample of glycogen from a patient with liver disease is incubated with Pi, normal glycogen phosphorylase and normal debranching enzyme. The ratio of glucose-1phosphate to glucose formed in this reaction mixture is 100. What is the patients likely enzymatic deficiency? What is the probable structure of the patients glucogen? Which of the following statements about the hormonal regulation of glycogen synthesis are correct? a) b) c) d) e) 6. Insulin increases the capacity of the liver to synthesize glycogen. Insulin is secreted in response to low levels of blood glucose. Glucagon and epinephrine have opposing effects on glycogen metabolism. Glucagon stimulates the breakdown of glycogen particularly in the liver. The effects of all three of the regulating hormones are mediated by cAMP.
5.
Place the following steps of the reaction cascade of glycogen metabolism in the proper sequence: A) b) c) d) e) Phosphorylation of protein kinase Formation off cyclic AMP by adenylate cyclase Phosphorylation of phosphorylase b Hormone binding to target cell receptors Phosphorylation of glycogen synthase a and phosphorylase kinase
7. 8.
What is the ultimate source of most of the essential amino acids required by humans? Which of the following are intermediates in the pathway for the biosynthesis of both phenylalanine and tryptophan? a) b) c) d) Anthranilate Chorismate Shikimate Prephenate
p80
9. 10. 11. 12. 13.
What are the intermediates in the flow of nitrogen from N2 to heme (i.e. indicate at which intermediates nitrogen atoms are exchanged)? Methotrxate, a folate antagonist, interferes with nucleic acid biosynthesis in bacteria. Would you expect it to inhibit purine or pyrimidine biosythesis? Explain. Why might covalently linked enzymes, such as those of the pyrimidine biosynthetic pathway of mammals, be advantageous to an organism? What is the committed step in pyrimidine biosynthesis? Biosynthetic pathways that require NADPH include which of the following? a) b) c) d) e) Gluconeogenesis Fatty acid biosynthesis Ketone body formation Cholesterol biosynthesis Tyrosine biosynthesis
14.
Suppose that genetic engineering techniques enable you to transfer the genes for the enzymes of the glyoxalate pathway to human tissues and to express them in mitochondria. Why might those wishing to lose weight rapidly be interested in such a system?
ANSWERS
1. Glycogen molecules have 1 reducing end. The number of nonreducing ends will be affected by the number of glucoses and the number of branch points, but there will always be only one reducing end. Muscle does not have glucose 6-phosphatase, the enzyme that catalyzes the conversion of glucose-6 phosphate into glucose. Therefore, muscle cannot produce glucose for export into the bloodstream. Muscle stores glycogen only for its own use. The phosphorolytic cleavage of glycogen produces glucose 1-phosphates. Hydrolytic cleavage produces glucose. Glucose 1-phosphate can be converted to glucose 6phosphate without using ATP. Conversely, glucose (produced from the hydrolytic cleavage) requires ATP for conversion to glucose 6-phosphate. Thus, glucose 1phosphate avoids an ATP consuming step upon entering glycolysis. glucose pyruvate
2.
3.
uses 2 ATP, produces 4 ATP = net gain of 2 ATP glucose 1-P pyruvate
p81
uses 1 ATP, produces 4 ATP = net gain of 3 ATP 4. The patient has a ratio of 100 glucose 1-P/ glucose upon breakdown of the glycogen. Normally the ratio would be about 11.5 glucose 1-P/ glucose (see notes: lecture 28, pg 5 at the top) because glycogen is frequently branched. The glucose residue at the branch point is given off as glucose; all of the rest are released at glucose 1-P. The patient has significantly fewer branches than normal. Their glycogen will not be as densely packed and will be longer. The extended shape of the molecule can cause damage to the cell, and may be responsible for the patients liver disease. The enzyme that is defective is probably the glycosyl-4,6 transferase (the branching enzyme used in glycogen synthesis). a, d d, b, a, e, c The essential amino acids in the diet of humans were ultimately derived from plants. b, c N2 NH4+ heme glutamate serine glycine -aminolevulinate porphobilinogen
5. 6. 7. 8. 9.
10.
Methotrexate is an analog of dihydrofolate (DHF) and is an inhibitor of dihydrofolate reductase (the enzyme that converts DHF to tetrahydrofolate (THF). The thymidylate synthase reaction converts N5,N10-methylene-THF into DHF during the process of methylating dUMP to dTMP. Inhibitors of dihyrofolate reductase prevent the conversion of DHF to THF. THF is required for both purine and pyrimidine metabolism, so both pathways would be affected. The clustering of two or more enzymes (or active sites on a single polypeptide chain) allows for efficient shuttling of products to the next enzyme for further synthesis. Also, the proximity of the enzymes minimizes side reactions because substrates are not allowed to diffuse away to other metabolic pathways. The formation of N-carbamoylaspartate by the aspartate transcarbamoylase (ATCase) is the committed step in pyrimidine biosynthesis. b, d, e In humans, fatty acids cannot get converted to carbohydrates (they arent glucogenic). Humans synthesize glucose from amino acids during non-fed times. If the enzymes of the glyoxalate pathway were introduced into humans, we would be able to make glucose from the carbons of fats instead of proteins. During times of hunger, we could break down fat instead of protein for conversion to glucose. This would result in quicker weight loss.
11.
12. 13. 14.
p82
Name___________________________________ Biochemistry 501 Third Exam---Question Packet READ THESE INSTRUCTIONS: Fill in your name and id # on the Scantron form now. Only the Scantron form will be graded and collected. All answers must be entered legibly by filling in the appropriate circle on the Scantron form with a #2 pencil. Each question is worth 3 points. You have 90 minutes to complete the exam. 1. In the liver of a well-fed adult, the primary function of the pentose phosphate pathway is: A. B. C. D. E. 2. to oxidize glucose to produce NADH to convert pentose phosphates to metabolic intermediates for oxidative phosphorylation to synthesize NADPH and pentose phosphates to synthesize sterols for reductive biosynthesis
Which of the following pathways require NADPH? I) fatty acid biosynthesis II) cholesterol biosynthesis III) fatty acid oxidation A. B. C. D. E. only I only II only III only I and II I, II, and III
3.
In the glutathione reductase reaction shown below, what is the molecule that gets oxidized (X)? G-S-S-G + X a. b. c. d. e. Glucose-6-phosphate NADPH ATP H2 O O2 2GSH + Y
p83
4.
Before fatty acid synthesis can occur, ______ is converted to ________ in the mitochondria and then is shuttled to the cytoplasm where it is cleaved by __________ to oxaloacetate and ______. A. B. C. D. E. pyruvate, Acetyl-CoA, pyruvate dehydrogenase, pyruvate acetyl-CoA, citrate, citrate lyase, acetyl-CoA lactate, Pyruvate, lactate dehydrogenase, glucose-6-phosphate acetyl-CoA, citrate, succinate dehydrogenase, Acetyl-CoA acetyl-CoA, malate, malate dehydrogenase, pyruvate
5.
Which of the following statements about acetyl-CoA carboxylase is INCORRECT? A. B. C. D. E. It catalyzes the rate-limiting step in fatty acid synthesis. It requires a biotin cofactor. It is part of the fatty acid synthase complex. It requires ATP. The product of the reaction is malonyl-CoA.
6.
Which of the following enzymes, substrates, or intermediates is NOT involved in the synthesis of palmitate or palmitic acid? A. B. C. D. E. mevalonate ATP fatty acid synthase acyl carrier protein NADPH
7.
The synthesis of cholesterol needs all of the following molecules except A. B. C. D. E. O2 acetyl-CoA ATP NADPH acetone
8.
Which of the following enzymes controls the rate of cholesterol synthesis? A. B. C. D. E. acetyl-CoA carboxylase ATP-citrate lyase lipoprotein lipase glycerol 3-phosphate dehydrogenase HMG-CoA reductase
p84
9.
Which component of LDL is recognized by the LDL receptor? A. B. C. D. E. Cholesterol Triacylglycerols ApoB-100 ApoC-II Phosphatidylcholine
10.
High Density Lipoprotein (HDL) functions to carry good cholesterol by A. B. C. D. E. Carrying endogenously derived cholesterol and triacylglycerides from the liver to the adipose tissue and muscle where they are stored or used for energy. Transporting unused cholesterol to the extrahepatic tissues where it can be packaged into HDLs for excretion from the organism. Carrying dietary cholesterol, triacylglycerides, and other lipids from the intestines to other tissues Transporting cholesterol from extrahepatic tissues back to the liver where it can be converted to bile salts Both C and D.
11.
Dietary cholesterol contributes towards high levels of LDL in blood by: A. B. C. D. E. down regulating LDL receptor synthesis. blocking the synthesis of acetyl-CoA carboxylase at the level of gene transcription. acting as a competitive inhibitor of VLDL transport. increasing levels of HDL. activating HMG-CoA reductase.
12.
Which of the following statements concerning steroid hormone receptors is INCORRECT? A. B. C. D. E. They are usually found in the cytoplasm or nuclei of cells. They contain a zinc atom in the DNA-binding domain. They contain a transcription activation domain. They activate the phosphoinositide signaling pathway. They are proteins.
13.
Which of the following statements about Vitamin D3 is INCORRECT. A. B. C. D. E. Vitamin D3 is a derivative of arachidonic acid and a precursor to a hormone essential in the regulation of pain and fever. Vitamin D3 is produced in the skin in a photochemical reaction driven by UV light Deficiency of Vitamin D3 leads to the formation of weak bones, resulting in the disease rickets. The active form of Vitamin D3 activates gene expression by binding to vitamin D3 receptor. Vitamin D3 is abundant in fish oils and is added to commercial milk as a nutritional supplement.
p85
14.
Which of the following is NOT a step in the phosphoinositide cascade? hormone binds a cell surface receptor occupied receptor causes GTP-GDP exchange activated PLC cleaves PIP2 to DAG and IP3 DAG binds a receptor in the endoplasmic reticulum, which releases Ca2+ and activates protein kinase C E. phosphorylation of cellular proteins by protein kinase C produces cellular response A. B C D
15.
Which statement concerning regulation of blood glucose levels is INCORRECT? A. B. C. D. E. Under conditions of high blood sugar, the cells of the pancreas secrete insulin Diabetics typically have low blood sugar levels because they excrete sugar in their urine Many Type II diabetics (non-insulin dependent) may have normal or even high insulin levels in their blood but defective insulin receptors Under conditions of high blood sugar, glycogen synthase is activated The process of glucose homeostasis is related to the fact that the brain requires glucose
16.
Which molecule is not an intermediate in gluconeogenesis? A B C D E Fructose 6 phosphate Fructose 1-6 bisphosphate Fructose 2-6 bisphosphate Glucose 6 phosphate dihydroxyacetone phosphate (DHAP)
17.
Which of the following statements about gluconeogenesis is FALSE? A B C D E. It is used to maintain a constant blood glucose concentration It uses non-hexose precursors to synthesize glucose Diabetes leads to accelerated gluconeogenesis Fasting leads to accelerated gluconeogenesis It, occurs only in plants
18.
The energy required to fuel gluconeogenesis comes from: A. B. C. D. E. Glycolysis -oxidation of fatty acids Glycogen breakdown Amino acid breakdown Fatty acid synthesis
p86
19.
Which of the following events would not cause an increase in blood glucose? A B C D E High cAMP High glucagon High epinephrine High insulin Activated Protein Kinase A
20.
How many net ATPs can be generated from glycolysis from one glycogen breakdown product and why? A. B C D E 3 ATP, the first activation step of glycolysis is bypassed 2 ATP, it is at the same state as glucose 4 ATP, it gets extra energy from breakdown 5 ATP, it starts at the payoff phase 1 ATP, it requires more energy for proper activation
21.
If a glycogen molecule has only 5 branch points, it has: A B C D E. 5 non-reducing ends and 1 reducing end 1 non-reducing end and 5 reducing ends 6 non-reducing ends and 1 reducing end 1 non-reducing end and 6 reducing ends 25 non-reducing and 25 reducing ends
22.
What does glycogenin do? A. B. C. D. E. primes the synthesis of glycogen via a Tyrosine residue primes the synthesis of glycogen via an Alanine residue It adds glucose residues to the reducing end of glycogen It forms UDP-glucose B and C
23.
Insulin regulates glucose utilization by: A. B. C. D. E. mediating transport of glucose from plasma across the plasma membranes of skeletal muscle, heart and adipose tissue increasing the export of glucose from muscle interacting with the glucagon receptor increasing cAMP levels activation of glucose 6-phosphatase in the liver
p87
24.
How does a glucose tolerance test work? A. B. C. D. E. A patient is given glucose solution, then is tested for blood glucose levels A patient is injected with epinephrine, and then tested for blood glucose levels A patient is injected with glucagon, and then tested for blood glucose levels A patient is injected with ATP and then tested for insulin levels A patient is given glucose solution, then is tested for blood albumin levels
25.
Glucagon regulates the rates of glycolysis and gluconeogenesis through Fructose-2,6bisphosphate (F-2,6-b-P) by which of the following ways: A. B. C. D. E. signaling an increase in PFK2 activity, increasing glycolysis signaling an increase in FBPase2 activity, increasing gluconeogenesis signaling an increase in FBPase2 activity, increasing glycolysis signaling a decrease in FBPase2 activity, increasing gluconeogenesis preventing the binding of insulin, leading to an inactivation of the insulin receptor pathway
26.
Which of the following pathway(s) contribute intermediates for the formation of amino acids? A. B. C. D. E. Citric Acid Cycle -Oxidation of fatty acids Pentose Phosphate Pathway A and B A and C
27.
The first step of porphyrin synthesis in animals starts with: A. B. C. D. E. acetyl-CoA and glycine malonyl-CoA and succinyl-CoA glycine and succinyl-CoA succinyl-CoA and oxaloacetate glutamate and glutamyl-tRNA
28.
The plant hormone indole-3-acetate (auxin) is formed from: A. B. C. D. E. Tryptophan Histidine Arginine Threonine Phenylalanine
p88
29.
Nucleotides are composed of: A. B. C. D. E. A hexose sugar and a phosphate A sugar, a fatty acid and a glycerol backbone A nitrogenous base and a sugar A nitrogenous base, a pentose sugar, and a phosphate A nitrogenous base, a hexose sugar, a phosphate, and DAG
30.
Which of the following is not a substrate for Ribonucleotide reductatse? A. B. C. D. E. UDP ADP CDP GDP dTDP
31.
In general, biosynthetic reactions are: A. B. C. D. E. oxidative and energy requiring. reductive and energy yielding. simple reversals of the catabolic pathways. completely independent of catabolic pathways. reductive and energy requiring.
. 32. Which of the following is NOT a type of allosteric regulation? A. B. C. D. E. 33. concerted feedback inhibition sequential feedback inhibition cumulative feedback inhibition feed forward activation phosphorylation
One of the following metabolic pathways occurs both in the cytoplasm and mitochondria: A. B. C. D. E. glycogen synthesis. gluconeogenesis TCA. fatty acid biosynthesis nucleotide biosynthesis.
p89
Answer Key: 1. D 6. A 11. A 16. C 21. C 26. E 31. E 2. D 7. E 12. D 17. E 22. A 27. C 32. E 3. B 8. E 13. A 18. B 23. A 28. A 33. B 4. B 9. C 14. D 19. D 24. A 29. D 5. C 10. D 15. B 20. A 25. B 30. E
p90

501 PartIII Spr2012

Hochgeladen von

Dokumentinformationen

Originalbeschreibung:

Originaltitel

Copyright

Verfügbare Formate

Dieses Dokument teilen

Dokument teilen oder einbetten

Freigabeoptionen

Stufen Sie dieses Dokument als nützlich ein?

Sind diese Inhalte unangemessen?

Copyright:

Verfügbare Formate

501 PartIII Spr2012

Hochgeladen von

Copyright:

Verfügbare Formate

Biochemistry 501 Spring 2012 Part 3 Course Packet

Lecture Topic Pentose phosphate pathway

Fifth Edition Pgs 558-563

Fourth Edition Pgs 549-555

Synthesis of fatty acids and phospholipids

Pgs 805-817; 820-831

Pgs 787-798; 808-815

Synthesis of ketones, sterols and isoprenoids

Pgs 666-668, 831-836

Pgs 650-652; 815-820

Cholesterol regulation, lipoproteins

Biological activities of lipids

Pgs 357-369; 817-820; 905, 908, 1144

Pgs 357-363; 800-803; 885; 888-889; 1109

Regulation of blood glucose I: Gluconeogenesis

Regulation of blood glucose II: Glycogen metabolism

Regulation of blood glucose III: Hormonal control

Biosynthesis of amino acids and porphyrins

Pgs 860-878; 909

Pgs 840-862; 889

Parts I, II and III

Parts I, II, and III

3 Options for Glucose-6-P

Pentose Phosphate Pathway

Glycogen Pyruvate Glycogen! Glycolysis!

Ribose! 5-phosphate Pentose Shunt!

Roles of Pentose Phosphate Pathway

Overview of Pentose Phosphate Pathway

3 Glucose-6-P + 6 NADP+ + 3 H2O ! 6 NADPH + 6 H+ + 3CO2 + 2 Fructose-6-P + Glyceraldehyde-3-P

Operates in two phases:

Pentose Phosphate Pathway

Overview of Pentose Phosphate Pathway

OH! H! OH! OH! CO2! ! ! H! H!

CH2OH! !! O! OH! OH!

3 Glucose-6-P + 6 NADP+ + 3 H2O ! 6 NADPH + 6 H+ + 3CO2 + 2 Fructose-6-P + Glyceraldehyde-3-P

Operates in two phases:

Total for oxidative reactions: two NADPH per glucose

Carbon skeleton rearrangements in the Pentose Phosphate Pathway

C H2OPO32 Ribulose-5phosphate (Ru5P)

C5!+!C5! ! ! ! ! C7 +!C3! ! ! ! ! C5 +!C4! ! ! ! ! Sum:! 3 C5! ! !

C3 +!C7! ! ! ! ! C6 +!C4! ! ! ! ! C6 +!C3! ! ! ! ! 2C6 +!1C3! ! ! ! ! !

Transketolase! Transaldolase! Transketolase!

C H2 OPO32 2,3-Enediolate intermediate

C H2OPO32 1,2-Enediolate intermediate

C H2OPO32 Xylulose-5phosphate (Xu5P)!

C H2OPO32 Ribose-5phosphate (R5P)!

First Transketolase Reaction

Production of Sedoheptulose-7-phosphate and Glyceraldehyde-3-phosphate

! OH! ! C! O! HO! C! H! + H! C! OH! CH! O P! 2 !

! OH! ! ! O! C! H! C! OH! C! OH! C! OH! CH! O P! 2 !

!! ! ! O! HO! C! H! H! C! OH! H! C! OH! +! H! C! OH! CH! O P! 2 ! Sedoheptulose-! 7-phosphate!

Second Transketolase Reaction

Flux of Metabolites through PPP

Modes of Pentose Phosphate Pathway

Modes of Pentose Phosphate Pathway

Mode 1: Nucleotide synthesis

NADPH! NADPH + CO2!

Modes of Pentose Phosphate Pathway

Regulation of Pentose Phosphate Pathway

TCA cycle CO2

Rate-limiting step: G-6-P dehydrogenase Rate is controlled by substrate availability: NADP+

Reactive Oxygen Species