ENZYMOLOGY
can catalyze the conversion of substrate to
1. Enzymes are organic catalysts
Organic found in living things
Catalyst fasten speed reactions
Enzymes are:
o Never consumed
o Never converted to products
o Maybe recovered or reused for future
reactions
2. Enzymes are affected by extreme temperature
At 56C enzymes undergoes denaturation
o Protein in nature
At 60C undergoes precipitation
3. The best temperature to measure enzyme is at
37C
Human body temperature
For every 10C rise in temperature, the:
o Reaction velocity is doubled
o Reaction time is cut into half
4. Michaelis Menten curve states the relationship
between reaction velocity and the concentration of
substrate
First (1st) Order Reaction
- If the concentration of the reaction is doubled,
the velocity of the reaction is doubled; (2/2)
Second (2nd) Order Reaction
- If the concentration of the reaction is doubled,
the velocity is quadrupled; (4/2)
Zero (0) Order Reaction
- Any increase in the concentration of the
substrate, there is no change in the velocity or
speed of the reaction
5. Serum is the specimen used in the measurement of
enzyme activity
In enzyme measurement, the activity of the
enzyme in serum is easier to measure
6. The units of enzyme are IU and katal
A. IU (International Unit)
Most common unit of enzyme activity
Refers to the activity of enzyme in serum that
can catalyze the conversion of substrate to
product per minute (m/m)
B. Katal
Refers to the activity of enzyme in serum that
product per second (m/s)
7. The approaches to enzyme activity includes the
Fixed-End Point Assay and the Kinetic Assay
2 approaches to Enzyme Activity in the serum
A. Fixed End Point Assay
Use of substrate and co-enzyme
Substrate enz in serum end products
that will act on substrate
End product + DNPH color rgt. end products
(dinitrophenylydrazine)
i.e. REITMAN FRANKEL METHOD
1. ALT or SGPT
Alanine and -ketoglutarate will be acted upon
by ALT to produce pyruvic acid and glutamic
acid
ALT is a transferase which catalyzes the transfer
of an amino group from Alanine to -
ketoglutarate
o Substrates: Alanine and -ketoglutarate
o Donor molecule: Alanine
o Recipient molecule: -ketoglutarate
o Co-factor used: Vitamin B6 or
pyridoxine/pyridoxal phosphate
ALT/ SGPT
Alanine + -ketoglutarate pyruvic acid + glutamic acid
Pyruvic acid + DNPH color rgt. Brown product
(dinitrophenylydrazine)
2. AST or SGOT
Aspartate and -ketoglutarate will be acted upon
by AST to produce oxaloacetic acid and
glutamic acid
AST is a transferase which catalyzes the transfer
of an amino group from Aspartate to -
ketoglutarate
o Substrates: Aspartate and -ketoglutarate
o Donor molecule: Aspartate
o Recipient molecule: -ketoglutarate
 o Co-factor used: Vitamin B6 or Aspartate and -ketoglutarate will be acted upon
pyridoxine/pyridoxal phosphate by AST to produce oxaloacetic acid and
glutamic acid
AST is a transferase which catalyzes the transfer
AST/ SGOT
Aspartate + -ketoglutarate O.A. acid + glutamic acid of an amino group from Aspartate to -
ketoglutarate
Oxaloacetic acid reacted upon by MDH (malate
Oxaloacetic acid + DNPH color rgt. Brown product dehydrogenase) to convert it to Malate
(dinitrophenylydrazine) Consequently, NADH (colored) will be NAD
(colorless). There is a removal of H+ from
B. Kinetic Assay
Monitors the absorbance of products many times NADH as gradual process.
o Substrates: Aspartate and -ketoglutarate
from the start to end of reaction at regular
o Donor molecule: Aspartate
interval the obtain average speed
o Recipient molecule: -ketoglutarate
Value submitted to the doctor
Uses 2 enzymes (coupled enzyme assay) AST/ SGOT
o ALT pairs with LD (lactate dehydrogenase) Aspartate + -ketoglutarate O.A. acid + glutamic acid
o AST pairs with MDH (malate
dehydrogenase) Oxaloacetic acid +NADH MDH Malate + NAD
Process is slow so choose speed that is constant
8. There are six classes of enzymes.
Substrate enz in serum end products Oxidoreductases, Transferases, Hydrolases,
that will act on substrate Lyases, Isomerases, and Ligases (OTHLIL)
A. OXIDOREDUCTASE
Class 1
End product + co-enzyme read the Abs with intervals Found in serum
Catalyzes oxidation and reduction reactions
i.e. KARMEN METHOD
(Redox)
1. ALT or SGPT Subclass:
Alanine and -ketoglutarate will be acted upon
o Oxidase
by ALT to produce pyruvic acid and glutamic o Dehydrogenase
acid Lactate Dehydrogenase (LD)
ALT is a transferase which catalyzes the transfer Malate Dehydrogenase (MD)
of an amino group from Alanine to - Isocitrate Dehydrogenase (ICD)
ketoglutarate Glucose-6-Phosphate Dehydrogenase
Pyruvic acid reacted upon by LD (lactate
(G6PD)
dehydrogenase) to convert it to Lactate Hydroxybutyric Dehydrogenase (HBD)
Consequently, NADH (colored) will be NAD o Oxygenase
(colorless). There is a removal of H+ from o Peroxidase
NADH as gradual process
o Substrates: Alanine and -ketoglutarate B. TRANSFERASE
o Donor molecule: Alanine Class 2
o Recipient molecule: -ketoglutarate Found in serum
Involves in transferring of amino group
o Transaminase
ALT/ SGPT Serum Glutamate Oxaloacetic acid
Alanine + -ketoglutarate pyruvic acid + glutamic acid
Transaminase (SGOT)
Aspartate Transferase (AST)
Pyruvic acid +NADH LD Lactate + NAD Serum Glutamate Pyruvic acid
2. AST or SGOT Transaminase (SGPT)
 Alanine Transferase (ALT) 1st: substrate
o Transpeptidase 2nd: reaction catalyzed
Gamma-Glutamyl Transpeptidase (GGTP) 3rd: co-enzyme
Gamma-glutmayltransferase (GGT) o i.e. Alanine tranferase
o Phosphotransferase C. IUB NAMING
Kinase International Union of Biochemistry Number-
C. HYDROLASE name of Enzymes
Class 3 1st: class (ex. Oxidoreductase)
Found in serum 2nd: subclass (ex. Oxidase)
o Amylase (AMS) 3rd: subclass (ex. Dehydrogenase)
Hydrolysis of Amylum (Starch) substrate 4th: serial number (specific)
o Lipase (LPS) o i.e. EC1.1.1.27
Hydrolysis of Lipids substrate
In general: Olive oil 10. Enzymes with isoenzymes are ACP, CK, ALP, and
o Cholinesterase (CHS) LD
Hydrolysis of Acetylcholine - substrate Catalyzes the same reaction
o Phosphatase o Iso same
Hydrolysis of organic Phosphate Esters - o Enzyme reaction
substrate Multiple form of enzymes
p Nitrophenyl phosphate There is slight variation in molecular structure
Glycerophosphate and physical properties
Thymolphthalein monophosphate Can be separated by electric current via
Acid Phosphatase (ACP) active at pH 5 Electrophoresis
Alkaline Phosphatase (ALP) active at pH Enzymes with isoenzymes: ACP, CK, ALP, LD
9.8 I. ACID PHOSPHATASE: 2 isoenzymes
a. Inside prostate
D. LYASE
PAP: Prostatic acid phosphate (70% activity)
Class 4
Normally present in Prostate gland
Not found in serum
Should be within reference range in serum
Involves in removal of functional group
If abnormally elevated:
o Aldolase
Substrate: Fructose 1,6 diphosphate o Prostate cancer/ prostatic cancer/ prostatic
carcinoma
E. ISOMERASE b. Inside RBCs
Class 5 RBC acid phosphate (30% activity)
Not found in serum Normally present in RBC
Involves in interconversion Should be within reference range in serum
F. LIGASE If abnormally elevated:
Class 6 o Hemolytic Transfusion Reaction (HTR)
Not found in serum
Involves in joining of cut parts 2 ways of separating the isoenzyme of ACP

1. Electrophoresis
9. Naming of enzymes may done by trivial naming, Dye the different bands and scan
scientific naming, and IUB naming
eletrophoregram using Densitometer
A. TRIVIAL NAMING Fast Migrating PAP
Name from substrate Slow Migrating rbc ACP
o i.e. Amylum: Amylase, Lipid: Lipase Other isoenzymes close to the band of PAP:
B. SCIENTIFIC NAMING o ACP rom granulocytes
2 or 3 parts o ACP from platelets
o 1st and 2nd are frequently used o ACP from the monocytes
 2. Chemical Inhibition Technique IV. LACTATE DEHYDROGENASE
Uses chemicals Has 5 isoenymes
o Cupric ion (Cu2+) But sometimes 6 isoenyzmes, if patient has
o Tartrate (C4H4O6-2) chronic alcoholism
a. PAP o LD6 Alcohol Dehydrogenase
Cu2+ - No Effect o Minimal
Tartrate Inhibited LD is a tetramer; it has 4 subunits
b. Rbc ACP Separated by electrophoresis
Cu2+ Inhibited Normally in the serum, the order from highest
Tartrate No Effect activity to lowest activity:
II. CREATINE KINASE: 3 isoenyzmes o LD2 > LD1 > LD3 > LD4 > LD5
Old: Creatinine Phosphokinase o Ratio of LD1/LD2 = less than 1 (0.5 0.75)
CK is a dimer; it has 2 subunits: In myocardial infarction, both LD1 and LD2
o B subunit are elevated (but LD1 is higher than LD1)
o M subunit o Ratio of LD1/LD2 = greater than 1
In electrophoresis, the numbers are based on o Flipped Ratio
the order of decreasing rate of migration 1. LDH4
The 3 isoenzymes are stained with dye and are LD1
scanned by densitometer Abundant in the heart
a. CKBB (Brain) Fastest migrating
Almost absent in the serum because of the Elevated in myocardial infarction
Blood-Brain barrier 2. LDH3M
In severe head injury affecting the BBB: CKB LD2
is present in elevated concentration in serum Abundant in the heart
CK1; fastest migrating Elevated in myocardial infarction
b. CKMB (Heart) 3. LDH2M2
From myocardial tissue From the lungs
Elevated in myocardial infarction LD3
CK2 4. LDHM3
c. CKMM (Muscle) From the muscles and liver
Also elevated in myocardial infarction LD4
CK3 Elevated in muscle dystrophy and liver damage
5. LDHM4
III. ALKALINE PHOSPHATASE LD5
Widely distributed; from many sources; many From the muscles and liver
isoenzymes Elevated in muscle dystrophy and liver damage
Also used as a tumor marker
o Regan Isoenyzme Lung cancer 11. Enzymes are organ specific
o Kasahara Isoenzyme GI cancer Prostate: ACP (specifically PAP)
o Nagao Isoenzyme - Adenocarcinoma Semen: ACP (specifically PAP)
a. Liver ALP Pancreas: AMS, LPS
Fast Liver ALP Heart: CK, AST/SGOT, LD
Slow Liver ALP Liver: ALP, ALT/SGPT, LD, GGT
b. Bone and Placental ALP Bone: ALP
Co migrators Determination of Prostatic Cancer
Bone ALP: Thermolabile (destroyed by heat) ACP (PAP)
Placental ALP: Thermostable Determination of Rape
o Reduced activity with Phenylalanine ACP (PAP)
c. Intestinal ALP Determination of Heart Attack
Elevates during meal
CK, AST, LD
 Determination of Liver Damage Starch
ALP, ALT, LD o Amyloclastic method
Determination of Chronic Alcoholism Maltose
GGT o Saccharogenic method
Determination of Pesticide/Insecticide Glucose
Poisoning o Somogyi method
CHS B. LIPASE
Determination of Pancreatitis a. Turbidimetric method
AMS, LPS Aka Cherry Crandall method
Disappearance of Triglycerides
12. Enzymes act on their substrates which are specific b. Titrimetric method
in nature Titration of Fatty Acids by standard alkaline
Substrate of ACP/ALP: Organic Phosphate Esters c. Colorimetric method
o Glycerophosphate Colors glycerol
Bodansky method
o P-Nitrophenyl Phosphate Chromotrophic acid
King Armstrong method HCHO Pink Chromophore
o Thymolphthalein Monophosphate Color rgt.
Roy method
Substrate of AST: Aspartate/Aspartic Acid
Creatinine loss of H2O Creatine
Substrate of ALT: Alanine
Substrate of GGT: Gamma glutamyl
Substrate of CHS: Acetylcholine 3 ENZYMES FOR HEART DIAGNOSIS
o True CHS substrate ACh from human body
o Pseudo CHS substrate butyrylthiocholine 1. Creatinine Kinase (CK)
Amylase: Starch Tanzen Givarg method
Lipase: Lipids Oliver Rosalki mehod
2. Aspartate Transferase (AST)
13. The enzyme are measured in the laboratory by Reitman Frankel method (FEP approach)
mostly color reaction Karmen method (Kinetic approach)
3. Lactate Dehydrogenase (LD)
A. AMYLASE
Wacker method
2 isoenzymes
Wroblewski Ladue method
o Salivary Amylase (S-AMS)
From salivary glands In Myocardial Infarction, proper order of:
o Pancreatic Amylase (P-AMS)
From pancreas a. Appearance
Only protein excreted in the urine CK: 4 6 hours after episode
o MW of AMS: 45,000 60,000 daltons AST: 6 8 hours after episode
o Pores of glomerulus in kidney: 60,000 daltons LD: 8 10 hours after episode
for filtration purposes b. Peak (best time to collect serum)
To diagnose pancreatitis with AMS: use both CK: 12 24 hours
serum and urine specimen AST: 48 hours
LD: 72 hours
AMS Maltase c. Disappearance
Starch Maltose Glucose CK: 1 2 days
AST: 4 5 days
LD: 7 12 days
O4- (periodate)
Glycerol Formaldehyde (HCHO) Myocardial Tissue Protein
Oxidize
1. Myoglobin
o Start-up in 2 3 hours
2. Treponin
o TnC Calcium unit
o TnI Inhibitory unit
o TnT Tropomyosin unit
*measured in laboratory (2): TnI and TnT
OTHER ENZYMES DISCUSSED:
LAP Leucine aminopeptidase
5 NT 5 nucleotidase
o Test for hepatic damage, specifically biliary
tree/duct
HBD Hydroxybutyrate Dehydrogenase
o For myocardial infarction