How does a cell fine tune the activity of its enzymes?

9/12
How do cells control enzyme activity?
What is allosteric regulation? Can it stimulate or inhibit?
How does allosteric regulation control glycogen synthesis?
How can the products of a reaction pathway affect the enzymes that lead substrate
into the pathway?
What is the most common type of covalent modification?
How can proteolytic cleavage be used to regulate enzyme function?
What is a ribozyme?
What are the functions of a plasma membrane?



Assignment Due at start of class:
CH 5Que #: 5-6, 5-7
CH6 Que#: 6-1, 6-4, 6-5 and 6-6
Copy of answer key is located in
library at info desk for short term
check out. 10 pts
Assignment Due: CH Que # 5-6, 5-
7, 6-1, 6-4, 6-5 and 6-6)
Copy of answer key is located in
library at info desk for short term
check out. 10 pts
What are the ten different types of enzyme?
1) Hydrolase: cleaves substrate by adding H2O
2) Nuclease: cleaves adjacent nucleotides with H2O

3) Protease: cleaves peptide bonds with H2O
4) Synthase: makes complex molecule from precursors

5) Isomerase: rearranges functional group on substrate
6) Polymerase: synthesize DNA or RNA from nucleotides

7) Kinase: adds a phosphate to target
8) Phosphatase: removes phosphate from target

9) Oxido-Reductase (Dehydrogenase): one molecule is oxidized
and one reduced
10) ATPase: hydrolyzes ATP to ADP to generate energy

Some overlap may exist between types!


Lets Review the ways to control enzyme activity:
Which are available to prokaryotes?
Enzyme Production/Destruction
Via DNA/mRNA transcription/translation:
Release into the blood/target
Failure to replace it after turnover
Temperature and pH:
Allosteric Inhibition/Activation:
Catalytic/Regulatory subunits-
Covalent Modification:
Phosphorylation/Dephosphorylation-
Protein Kinase/Protein Phosphatase-
Proteolytic Cleavage:
Autocatalytic Activity:


How do cells control enzyme activity?
Some produce E only when needed (think prokaryotes!)
Some let E turn-over when no longer used
Some modify activity of E to meet needs at the time

FeedBack-Inhibition: Sometimes a product inhibits E to prevent
excess/wasteful production of product

FeedForward-Stimulation: Sometimes a product stimulates E to promote
greater production of product

Classic Pattern: molecule exerting effect on entry enzyme is often the
last molecule produced in the pathway!
A BCDE but E-inhibits/stimulates entry to pathway at the
AB-Enzyme
Allosteric regulation occurs when an enzyme exists in one of two
forms (fast/slow) and form taken is dependent upon the binding of a
regulator at a site other than the active site.
Feed Back Inhibition/ Allosteric Inhibition:
Observation about Glycolysis:
G + ATP G-6-P + ADP (Hexokinase)
Committed first step of glycolysis!

G-6-P can non-permanently bind Hexokinase at an allosteric site
and modify its conformation!

The Two Forms of Hexokinase:
Fast (no G-6-P) vs Slow(when G-6-P Present!)
+Km or +Vm -Km or -Vm
Why is this useful to a cell? What happens after a meal in a
diabetics skeletal muscle cell?
Allosteric regulation: enzyme exists in one of two forms (fast/slow)
and form taken is dependent upon binding of a regulator at a site
other than active site.
Feed FORWARD Stimulation/ Allosteric activation:
Observation regarding Glycolysis:
G-6-PF-6-PF-1,6-PPEP(Pyruvate Kinase)Pyruvate
How do we speed up the system at the END based on the presence of the
entry of substrate at the START of system?

Translated: how does a cell speed up energy use when there is plenty of energy
available?
Early product F-1,6-P Stimulates PK activity at last step!

The Two Forms of pyruvate kinase:
Fast vs Slow
When do you want these to be fast/slow?
Signal? F-1,6-P is allosteric regulator promoting FAST PK-form
Allosteric
Regulation of
an Enzyme
(protein):
Molecule binds
to non-active
site!
Typically
binding to
dimers or larger!
Often regulator
is product in
pathway!
Cells also regulate activity by covalently modifying enzymes by covalent
modification, typically adding/removing PO4-groups! Glycogen
synthase and Glycogen phosphorylase are reciprocally regulated classics
in this regard!
Glycogen is broken down to G-1-P either slowly or rapidly
depending on the activity state of Glycogen Phosphorylase a!
How do we know when to phosphorylate?
In this case phosphorylase kinase (a cAMP dependent protein kinase)
is able to phosphorylate its target glycogen phosphorylase a when the
hormone glucagon causes the cell to accumulate cyclic AMP.
cAMP made when cell is hungrybinds to..
cAMP-dependent Protein kinase
Phosphorylates targetactivation of enzyme!
Proteolytic cleavage can also be an important signal for enzyme
activation. This way an enzyme is not turned on until it is in the
correct location in body/cell.
Applications in the intestine and in Emphysema
Pancreatic Enzymes:
Zymogens: In active protein products
Pancreatic Zymogens:
Typsinogen, Chymotrypsinogen, Procarboxylpeptidase
Intestinal Enterokinase only cleaves Trypsinogen!
Trypsinogen activates remaining zymogens!

The liver also produces a plasma protein called:
Alpha-1-antitrypsin (irreversible competitive inhibitor) to prevent
inappropriate self-digestion of elastin and other connective fibers.
What happens to the balance of trypsin secretion and activity
in a chronic smoker? An irreversible competitive inhibitor is
lost! Trypsin is activated and digests collagen and elastin!
Lung loses all elasticity!
1) Smoking oxidizes a methionine residue on inhibitor!
R-CH2-CH2-S-Ch3 R-CH2-CH2-SO-CH3
2)Oxidized product cant inhibit enzyme!
Damaged lungs are rich in neutrophils
3) Trypsin is released and elastin destroyed!
4) Lungs must now forcibly contract to inhale AND exhale!
5) Our old male cadaver in the AP lab had this!
The pancreas produces a similar protein inhibitor to protect
itself against any trypsin that has its regulatory tail
removed accidentally!
Ribozymes (RNA) are the least common enzyme (they do not
consist of protein). They are typically autocatalytic in
function.
More about enzymes and kinetics 9/14
More about M-M kinetics hyperbolic curves, and approximations
LB plots, straight lines, and exact predictions
Why is it valuable to know km, Vm, V1/2, etc with respect to cell
culture or drug metabolism by cells?
Drugs as non-competitive and competitive inhibitors in cells
LAST MATERIAL FOR FRIDAYS EXAM:
Michealis-Menton Kinetics will be on exam
Lineweaver-Burke plots will not be on exam
50 points (bring scantron)
Exam will be Ch 1-3(mostly review) and 4-6
Exam will emphasize what is in the notes
Textbook should help understand the notes and is important,
especially the problems in the back of the chapters.



The active site is where the substrate binds to the
enzyme.
What are the six features of an active site?
1) AS is small part of total!
2) AS spatial in 3-D!
3) AS hold substrate by using
its specific charges on its
amino acids to attract
substrate!
Ionic, Hydrogen, Van der
Waals Forces
Covalent bonds only at
transition state!
4) AS located in peptide cleft!
5) Substrate/Cleft perfectly
align!
6) Substrate/Enzyme binding
causes induced fit at site!
Induced fit result in change to
shape of entire enzyme!
Competitive and Non-competitive Inhibitors bind to different sites!
Michealis-Menton Enzyme Kinetics refers to how fast
(quantity/time) an enzyme catalyzes a particular chemical
reaction! There are special rules that apply inside a cell!
Velocity: [P]/time!
Initial Reaction Velocity: speed at start!
Saturation: When an enzyme cant handle ANY more substrate per
time,,,all active sites are filled up!

Vmax: Maximum theoretical velocity!=traffic jam
Vmax: Half of maximum, typically organisms let their enzymes
function near this value.

Michaelis-Menton Kinetics: refers to the fact that an the substrate
MUST bind to the enzyme BEFORE becoming product! This creates
a special type of Keq Equation!
Normal: SubstrateProducts Keq=[p]/[s]
M-M Kinetics: Substrate + Enz >Enz-S<Product + Enzyme
There are now 4 reaction directions to consider in this reversible
reaction! Keq is now called Km or a Michaelis-Menton Constant!
Each enzyme has a specific Km value associated with it!
Km Values are important to cell biologists because they let us
know the health/unhealth of a cell given a set of conditions
and let us predict cell function/adaptability!
How much toxin/time can the cells of a weed degrade? When does
farmer Pat need to reapply herbicide and how much?

How much lactose can Pat digest/time? How much milk can Pat
drink without getting diarrhea, assuming lactose insufficiency?

How much chemotherapy drug can be destroyed/time by the
hapatocytes of a healthy or unhealthy liver? When does Bob need to
take more or less? What is the monetary cost?

How much glucose can be metabolized over time by the myocytes
of an Olympic sprinter? How much of his illegal drug can be
metabolized? Will he beat the test?
Competitive inhibitors change Km!-MM
Because fewer sites are available to the substrate!
Non-Competitive Inhibitors change Vm!-MM
Because fewer enzymes are available to the substrate!
Competitive and Non-competitive Inhibitors bind to different sites!
Irreversible Enzyme Inhibitors: DFP is similar to the Sarin
Nerve Gas for use on humans in warfare


Lineweaver-Burke Plots prepresent a double reciprocal system for
comparing reaction velocity to substrate concentration.
Competitive inhibitors change 1/Km!-LB
Because fewer sites are available to the substrate!
Non-Competitive Inhibitors change 1/Vm!-LB
Because fewer enzymes are available to the substrate!
Consider these problems:
1) With respect to 1/Km and 1/Vmax, which of these two Lineweaver-Burke Plots (See below) is from a system experiencing Competitive
and Non-Competitive Inhibition?
2) If you need the product of a reaction that is irreversibly inhibited (I.e. Sarin Nerve Gas effect on acetylcholinesterase druign warefare),
what must the cells of your body do to survive in the presence of the irreversible inhibitor?
LB Plot with inhibitors
Is A or B competitive and non-competitive?