Enzymes

Introduction
- two fundamental conditions for life:
1. the living entity must be able to self-replicate
2. the organism must be able to catalyze chemical reactions efficiently and selectively

Enzymes
- Are central to every biochemical process
- Acting in organized sequences, they catalyze the hundreds of stepwise reactions that degrade nutrient
molecules
- Conserve and transform chemical energy and make biological macromolecules from simple precursors

Biological catalysis
- was first recognized and described in the late 1700s, in studies on the digestion of meat by secretions of
the stomach
- in the 1800s with examinations of the conversion of starch to sugar by saliva and various plant extracts
- In the 1850s, Louis Pasteur concluded that fermentation of sugar into alcohol by yeast is catalyzed by
“ferments.”
- In 1897, Eduard Buchner discovered that yeast extracts could ferment sugar to alcohol, proving that
fermentation was promoted by molecules that continued to function when removed from cells
- Frederick W. Kühne called these molecules as enzymes
- The isolation and crystallization of urease by James Sumner in 1926 provided a breakthrough in early
enzyme studies
- Only in the 1930s was Sumner’s conclusion widely accepted, after John Northrop and Moses Kunitz
crystallized pepsin, trypsin,

Enzymes
- proteins which hasten biological catalysis without being consumed or altered during
the process known as organic catalyst
- measured in terms of their activity rather than absolute value.
- Importance: serves as catalyst in different biochemical reactions in the body

Properties of Enzymes
1. Protein in nature
2. Catalyze specific reaction
3. shows specificity with regards to substrate and effect.
4. Susceptible to denaturation
- extreme temperature
- extreme pH
- Irradiation
Uses of enzymes
A. Diagnostic tool
1. Myocardial Infraction (MI) - LDH, SGOT, CK/CPK
2. Liver profile (ALT, GGT)
3. Bone (ALP)
4. Prostate (ACP)
5. Pancreas (Lipase, Amylase)

B. Laboratory tests
- Hexokinase
- glucose
- Cholesteol esterase
- Cholesterol
- Lipase
- TAG

C. Treatment

General Principles of Enzyme Catalysis

Pattern of reaction of Enzyme

E + S ES E + P
Concepts on Enzyme-
Substrate
reaction
1. Fischer’s Lock and Key hypothesis
2. Koshland’s induced-fit hypothesis
3. Michaeles Menten concept
a. First order reaction
b. Zero order reaction

First order reaction

- the rate of reaction is determined by the concentration of substrate as well as enzyme
- the rate of reaction changes continuously with time as the substrate is consumed
Zero order
- the rate of reaction is linear with time, independent of concentration of substrate directly proportional
to enzyme concentration

Enzyme-Substrate Concentration Affects the Rate of Enyme-Catalyzed Reactions

 - This idea was expanded into a general of enzyme action, particularly by Leonor Michaelis and Maud
Menten in 1913.
- A key factor affecting the rate of a reaction catalyze by an enzyme is the concentration of substrate, [S].
- One simplifying approach in kinetics experiments is to measure the initial rate (or initial velocity),
designated V0, when [S] is much greater than the concentration of enzyme, [E].
- V0 can then be explored as a function of [S], which is adjusted by the investigator.
- The effect on V0 of varying [S] when the enzyme concentration is held Constant, at relatively low
concentrations of substrate, V0 increases almost linearly
- with an increase in [S]. At higher substrate concentrations, V0 increases by smaller and smaller amounts
in response to increases in [S].
- Finally, a point is reached beyond which increases in V0 are vanishingly small as [S] increases. This
plateau-like V0 region is close to the maximum velocity, Vmax.
- The ES complex is the key to understanding this kinetic behavior,

Factors affecting enzyme reaction

1. Substrate concentration
- specific for each particular enzyme
a. Follow the Michaeles Menten concept
b. E + S = ES complex
- rate of reaction is dependent on ES complex
- S conc., S bound to E, rate of rxn.
- when all E bounded to S, no further increase in reaction rate will be observed

2. Conditions necessary for chemical reaction - thermodynamically possible

3. Co-factors
- non-protein substances added in the enzyme-substrate complex before enzyme activity can
be manifested
a. Co-enzymes
- organic compound that hasten chemical reaction but undergo change or consumed to
another product (NAD, NADP)

Co-enzymes/vitamine precursor
- NAD (Niacin, Nicotinic acid)
- NADP (same with NAD)
- FMN (Riboflavin, Vit. B-2)
- FAD (same with FMN)
- Co-enzyme Q (Panthothenic acid)
- Co-enzyme A (same with Q)
- Pyridoxine PO-4 (Pyridoxine)

b. Activators
 - in-organic ions that helps binds to active site of enzymes by forming ionic bridges
- proper orientation of substrate to active site
- cat-ions: Mg, Mn, Fe, K, Ca
- an-ions: Cl, Br, NO3

c. Metalloenzymes - in-organic ions attached to a molecule

4. Inhibitors
- binds to the active site, blocking of access of Enzyme to Substrate
- occurs when reaction is proceeding at a rate less than expected due to some changes in pH,
temperature, and substrate
a. Competitive - binds to the active site of enzyme
b. Non-competitive - binds to other site of enzyme

5. Iso-enzymes
- enzymes with similar enzymatic activity but differ in physical, biochemical and immunological
properties
- have different specificities for other substrate

6. Physical/Chemical factors
1. Temperature
- optimum temp. 37
- activation of enzyme, 30
- freezing does not affect most enzymes

2. pH
 - each enzyme has optimum pH
- changes in O.P. Alters enzyme activity
- O.P. Occurs when sensitivity of measurement is maximal

Enzyme regulation
- The activities of metabolic pathways in cells are regulated by control of the activities of certain enzymes
- Keeps the production and utilization of each metabolic intermediate in balance

1. Feedback inhibition
- the regulatory enzyme is specifically inhibited by the end product of the pathway whenever the
concentration of the end product exceeds the cell’s requirements
- When the regulatory enzyme reaction is slowed, all subsequent enzymes operate at reduced
rates as their substrates are depleted
- The activity of allosteric enzymes is adjusted by reversible binding of a specific modulator to
regulatory site
- Modulators may be the substrate itself or some other metabolite, and the effect of the
modulator may be inhibitor or stimulatory
- The kinetic behavior of allosteric enzymes reflects cooperative interactions among enzyme sub-
units

e.g.
allosteric feedback inhibition of the bacterial enzyme system that catalyzes the conversion of L-
threonine to L- isoleucine in five steps.

- needed for protein synthesis, the

unused isoleucine accumulates
- and the increased concentration inhibits the catalytic activity of the first enzyme in the pathway
- immediately slowing the production of iso-leucine

2. Reversible Covalent Modification

- activity is modulated by covalent modification of the enzyme
molecule
- modifying groups include phosphoryl, adenylyl, uridylyl,
methyl, and adenosine diphosphate ribosyl groups
  Many proteolytic enzymes are synthesized as inactive precursors called zymogens, which are activated
by cleavage of small peptide fragments
 Enzymes at important metabolic intersections may be regulated by complex combinations of effectors,
allowing coordination of the activities of interconnected pathways

Means by which enzymes released in serum/plasma

1. During enzyme metabolism and excretion
2. Cells subjected to stress
3. Injured cells or cell death
a. Hypoxia f. Chemical/Drugs
b. Physical agents g. Microbial agents
c. Immune mechanisms
d. Nutritional disorders
e. Genetic defects

Classification of Enzymes in the blood

1. Plasma specific
a. Fibrinolytic
b. Thrombin
2. Secreted
a. Lipase b. Amylase
3. Cellular
a. Amino transferases
b. LDH
c. Phosphatase

Enzyme Nomenclature
- Catalogue with a four-digit Enzyme
- Commission number (EC number)
- The first digit indicates membership of one of the six major classes
- The next two indicate subclasses and sub-subclasses.
- The last digit indicates where the belongs in the sub-subclass

e. g.
Lactate Dehydrogenase
the EC number 1.1.1.27
(class 1, oxidoreductases; subclass 1.1,
CH–OH group as electron donor; sub-subclass 1.1.1, NAD(P)+ as electron acceptor).

Enzymes with similar reaction specificities are grouped into each of the six major
classes:
1. The Oxidoreductases (class 1)
- the transfer of reducing equivalents from one redox system to another
Oxidase: Cytochrome
Reductase: Dehydrogenase
1. ID 3. MD 5. G-6-PD
 2. LDH 4. ICD 6. HBD
2. The Transferases (class 2)
- catalyze the transfer of one molecule to the another
1. AST (SGOT)
2. ALT (SGPT)
3. CK (CPK)
4. GGT
5. OCT

3. The Hydrolases (class 3)

- are also involved in group transfer, but the acceptor is always a water molecule
Esterases
- ACP - CHS - ALP - LPS

Peptidases
- LAP - PPS - PTs

Glycosidases
- Amylase
- Glycoside
- Aymlo 1-6 glycosidases
- Galactosidases

4. Lyases (class 4)
- generally require coenzymes often also referred to as “synthases”
- catalyze reactions involving either the cleavage or formation of chemical bonds, with double
bonds either arising or disappearing
 ALD
 Glutamate decarboxylase
 Pyruvate decarboxylase
 Tryptophan decarboxylase

5. The Isomerases (class 5) move groups within a molecule, without changing the gross composition
of the substrate
- glucose phosphate isomerase
- ribose phosphate isomerase

6. The ligation reactions catalyzed by Ligases (“synthetases,” class 6) are energy-dependent and are
therefore always coupled to the hydrolysis of nucleoside Triphosphates
- bond formation between two groups of atoms with ATP as energy source
 Enzyme Determination
- measurement of amount of product formed per unit time
- measurement of amount of substrate per unit time

Interferences
1. Hemolysis
2. Lactescent serum
3. Storage (-20 degree Celsius / 2 -8)

Types of Enzyme Assay

1. End point assay
- reaction is allowed to proceed for a set of time after addition of substrate.
- no direct way to assess linearity of the reaction
- under-estimation in case of increase enzyme activity
2. Multi-point assay
- measures change in concentration at certain interval
- allow assessment of linearity of reaction
- some enzymes show Lag phase
 3. Kinetic assay
- involves the continuous measurement of change in concentration as function of time
- time consuming, impractical

Unit of Reporting
U/L or IU
- refers to the amount of substrate utilized or product produced in terms of umol/min/liter of blood and
other body fluids under control conditions
- recommended by IUB and IUPAC
 Specific Enzymes
Acid phosphatase
(E.C. 3.1.3.2)
- orthophosphoric monoester phosphohydrolase, acid optimum
- pH optimum of 5.6 (4.9 – 5.0)
- for patient suspected for prostatic cancer (possible metastatic bone involvement)
- principal sources: Erythrocytes
Prostate

Important isoenzymes
1. Red cell ACP - inhibited by Cupric ion and Formalin
2. Prostatic ACP - inhibited by L-tartrates ion

PSA
(Prostate Specific Antigen)
- related substance used as alternative diagnostic tool for ACP
- elevated in neoplastic and hyperplastic prostatic tissue

Methods
1. Bodansky (Betaglycerophosphate)
2. Ashinowara
3. Jones
4. Reinhart
5. King Armstrong (Phenyl phosphate)
6. Gutman and Gutman
7. Bessy Lowry and Brock (BLB) - p-nitrophenyl phosphate
8. Hillmann (alphanapthyl phosphate)
9. Babson Reed
10. Rietz and Guilbault - 4-methyl umbelliferone phosphate
11. TMP (Thymolpthalein phosphate)
12. Immunologic method (RIA)
13. Fishman and Lerner (Pheny lphosphate)

Commonly used method

1. BLB (N.V. 1 – 12 U/L)
2. TMP (N.V. 0.5 – 1.9 U/L)

Clinical Significance
1. Prostate carcinoma 3. Niemann Picks Disease 5. Myelocytic Leukemia
2. Hyperparathyroidism 4. Gauchers Disease
6. Others:
Malignant neoplasm of breast
Osteoporosis and other bone disease
ACP – useful also in forensic diagnosis of rape (ACP in semen)
Alkaline phosphatase
(E.C. 3.1.3.1)
- orthophosphoric monoester phosphohydrolase, alkaline optimum
- optimum pH, 10 (8.6-10)
- diagnosis of Liver and bone disease

Isoenzymes
1. Liver ALP
2. Bone ALP (normally increase in growing children)
3. Renal ALP
4. Placental ALP (Regan type) - found in tumors (carcinoplacental)
5. Intestinal ALP - increase 2-4 hours after fat rich meal

Methods
 1 to 5 methods of ACP
 BLB (PNPP)
 Huggins and Talalay (Phenolpthalein Di-phosphate)
 Moss (Alphanaphtyl Phosphate)
 Klein, Bason Reed (Buffered Phenolpthalein Phosphate)
 Bowers-Mc Comb (PNPP)

N.V. (using PNPP)

Male - 90 - 190 U/L
Female- 85 – 165 U/L

Clinical Significance (highly increased)

1. Bone disease (Pagets, osteogenic carcinoma)
2. Hepatobilliary diseases

Moderately increased
1. Infectious hepatitis
2. Fanconi’s syndrome
3. Primary/secondary hyperthyroidism
4. Third trimester of pregnancy - due to increase placental ALP

Amylase
(AMS)
- 1,4 alpha D-glucan, Glucanohydratase
- pH optimum, 6.9 – 7.0
- isoenzymes
- salivary amylase (ptyalin)
- pancreatic amylase

Sources: Salivary glands, ovaries, pancreas

Types
1. Alpha amylase (endoamylase) - human amylases
2. Beta amylase (exo-amylase) - plant and bacterial

Methods
1. Saccharogenic - measures the amount of reducing sugars produced by hydrolysis of starch
2. Amyloclastic - based on the measurement of decrease substrate concentration
3. Chromometric - measures the time required for complete hydrolysis of starch by amylase
4. Amylometric - measures the amount of starch hydrolyzed in a fix period of time

N. V. (method dependent)
Male - 30 – 220 U/L
Female - 15% higher than male value

Clinical Significance
1. Pancreatitis
2. Non-pancreatic (tumor of ovary/lungs)
- Hyper-amylasemia
- Hyper-amylasuria

Transaminases/Aminotransferases
a. Alanine amino Transferase (ALT)
E.C. 2.6.1.2/ formerly, SGPT
Sources: Liver, skeletal muscles, Heart

b. Aspartate amino Tranferase (AST)

E.C. 2.6.1.1/formerly SGOT
Sources: Liver, heart, kidney, RBC, skeletal muscle

Methods
1. Reitman –Frankel colorimetric assay
AST ALT
Substrate Aspartate/alpha ketoglutarate Alanine/alpha ketoglutarate

Color developer 2,4 Dinitrophenyl hydrazine 2,4 Dinitrophenyl hydrazine

Incubation 1 hour 30 minutes
End product Oxalo-acetate Pyruvate

2. Method of Karmen
- coupled enzyme activity
- measurement od absorbance of reduced NAD at 340 nm

3. Reaction with Diazonium salt

- Keto acid react with diazo compound
Pyridoxal-5’phosphate
- coenzyme in transaminases reaction
- carry amino acid from one group to another

Vitamin B-6 - one of the co-factor

N. V. (ALT) - less than 55 U/L

Clinical significance
1. Liver disease: Viral hepatitis, Carcinoma, Cirrhosis
2. Hepatic parynchemal disease
3. Used also in the screening of blood

N.V. (AST): 5 – 35 U/L

Clinical Significance
1. Increased shortly after M.I.
2. Progressive muscular dystrophy
3. Increase in some parynchemal disease

Lactate Dehydrogenase
(LDH), E.C 1.1.1.27
L-lactate: NAD Oxido-reductase
- catalyzes the reversible oxidation of lactate to pyruvate
- Pyruvate to lactate (pH 8.8 – 9.8)
- Lactate to Pyruvate (pH 7.4 – 7.8)
- Useful in monitoring MI, Liver and Hematologic disorders

Sources: Heart, Liver, Skeletal muscles, RBC, Platelets and Lymph nodes

Isoenzymes
 LDH-1 (H-4), LDH-2 (H-3M) - found in heart muscles, RBC, Renal cortex
 LDH-1 - fast moving (electrophoresis)
 LDH-2 - has the largest concentration
 LDH-3 (H-3M-2) - found in all body tissues
 LDH-4 (HM-3), LDH-5 (M-4) - found in skeletal muscles, liver, Ileum, skin
 LDH-5 - slow moving (electrophoresis)

Inhibitors
 Mercuric ions (reacts with thiol group)
 Borate and oxalate (compete lactate to binding site)

Methods
1. Wroblewski-LaDue
- use reverse reaction, NADH (co-substrate)
- kinetic assay (340 nm)
2. Wacker method - forward reaction
 3. Bergers-Broida
4. Kubowtz and Ott
5. Wroblewski-Cabaud

N.V.
P to L - 95 -200 U/L
L to P - 35 – 90 U/L

Clinical Significance
1. Myocardial Infarction (LDH-1 and 2)
2. Hepatic disease
3. Hematologic disorders (hemolysis)

Highly increased:
1. Myocarditis
2. Angina pectoris
3. Peri-carditis
4. Severe shock
5. Anoxia

Creatine Kinase/Creatine Phosphokinase (CPK)

E.C. 2.7.3.2
ATP: Creatine N-phosphotranferase
- catalyzes a reaction responsible for the formation of ATP ion the tissues
- catalyzes the reversible phosphorylation of creatine by ATP
- detects damages to myocardial and skeletal muscle tissues

Sources: Skeletal muscle, Brain, Smooth muscle, Heart

Isoenzymes
1. CK-1 (BB) - Brain
2. CK-2 (MB) - Heart muscle (30%)
3. CK-3 (MM) - Muscle (cardiac 70%)

Inhibitors: Ca, Mg (brain)

Activators: Cu, Zn, Isocitrate, Excess ADP Sulfhydryl compounds

Methods
1. Tanzer-Gilvarg Assay - forward reaction (Creatine to CP)
2. Oliver-Rosalki Assay
- reverse reaction (CP to Creatine)
- both are known as coupled enzyme assay
- both methods have final step of measuring the absorbance of NADH at 340 nm
- both methods require sulfhydryl compound as activator
- Sources: Cysteine, Glutathione, Cleland reagent (dithiothreitol)
3. Sax and Moore
 4. Rosalki and Hiss

N.V.
Male: up to 160 U/L
Female: up to 130 U/L

Clinical Significance
1. Myocardial infarction (CPK is first to elevate)
2. Muscle diseases (Muscular dystrophy)
3. Diseases of CNS
- cerebral Ischemia, Reye’s syndrome
- Acute cerebro-muscular disease
4. Hypothyroidism (pre-disposing factor to IHD)

Increase total Ck and CK-2

1. Angina pectoris
2. Tachycardia
3. Congestive cardiac failure
4. Myocarditis
5. Cardiogenic shock
6. Trauma following heart surgery
7. Percutaneous Transluminal Coronary Angioplasty (PTCA)

Enzyme profile
A. Cardiac profile
1. CPK (MB)
Increases: 4 – 6 hrs. after onset of MI
Peaks at: 18 – 20 hours

2. AST/SGOT
Increases: 4 – 6 hours after onset
Peaks at: 24 - 36 hours

3. LDH 1 and 2
Increases: 8 – 12 hours after onset
Peaks at: 48 – 60 hours

B. Pancreatic profile
1. Amylase
2. Lipase
3. Trypsin
C. Hepatic profile
1. ALT
2. AST
3. GGT
4. ALP