Enzymes 5.

Holoenzyme
Biologic proteins that catalyze biochemical reactions An active substance formed by combination of a co-
Not consumed or changed in composition enzyme and an apoenzyme.
Found in all body tissue (intracellular) and is in 6. Proenzyme or Zymogens
serum after cell injury An inactive enzyme precursor
E.g. Coagulation factors and digestive enzymes
7. Allosteric enzymes
Regulator of cellular processes, but not all enzymes
are allosteric.
Some can be allosteric provided that they are
composed of quaternary structures with two or
more protein chain containing the active sites and
regulatory sites (binding sites).
The substances that bind on the regulatory sites are
called Regulator
Function of Enzymes Two kinds of allosteric enzymes:
Hydration of Carbon Dioxide (respiration) 1. Homoallostery
Nerve Induction This is a cooperative substrate binding and
activation wherein substrate is a homotropic
Muscle Contraction
effector. Therefore the binding of substrate to
Nutrient Degradation (Digestion)
one active site alters the substrate binding
Growth and Reproduction
affinity and/or catalytic activity at other active
Energy Storage and Use
sites on the multimeric enzyme.
2. Heteroallostery
Components of an Enzyme
This merely involves the regulation by
1. Active Site
heterotropic effector molecules, which can be
A cavity of an enzyme where substrates bind and
positive (activation) or negative (inhibition).
undergo a chemical reaction.
These heterotropic effectors usually bind at a
2. Allosteric Site
site other than the active site. Furthermore,
A cavity other than the active site that binds
these effectors can can activate or inhibit the
regulatory (effector) molecules.
activity of an enzyme.

Terms associated with enzymes

Enzyme Classification and Nomenclature
1. Substrates
The system for classification of enzymes that also
Substances acted upon enzymes
serves as a basis for assigning code numbers to
Specific for each of their particular enzyme
them.
2. Cofactors
These code numbers, prefixed by EC, which are now
Non protein substances added in the enzyme
widely in use, contain four elements separated by
substrate complex to manifest the enzyme
points, with the following meaning as appearing in
activity
example: for Alcohol: NAD+oxidoreductase as EC
o Coenzyme or Prosthetic group
number is 1.1.1.1
An organic cofactor
o The first number shows to which of the six main
Nucleotide (E.g. NAD, NADP) and
divisions (classes) the enzyme belongs,
Vitamins
o The second figure indicates the subclass,
o Activator
o The third figure gives the sub-subclass,
An inorganic cofactor
o The fourth figure is the serial number of the
Metal ion (E.g. Cl-1, Mg++,Cu+)
enzyme in its sub-subclass.
3. Isoenzyme
Similar enzymatic activity but differ in physical,
biochemical and immunologic characteristics
4. Apoenzyme
The protein portion of the enzyme
Subject to denaturation, in which enzyme losses its
activity
CLASS RECOMMENDE ABBREVIATED E.C SCIENTIFIC NAME 6. Ligases
D NAME NAME CODE
Catalyze the joining of two substrate molecules,
NO.
1. Oxido Lactate LDH 1.1.1.27 L-Lactate NAD+ coupled with breaking of pyrophosphate bond in
reductase dehydrogenase oxidoreductase ATP
2. 2.1 Aspartate SGOT ( Serum 2.6.1.1 L-Aspartate ,2- Ab + C AC + b
Transferase amino Glutamate oxaloglutarate E.g. Glutathione Synthetase (GSH-S)
transferase Oxaloacetate Amino transferase
transaminase)
ENZYME MECHANISMS
2.2 Alanine SGPT ( Serum 2.6.1.2 L-Alanine, 2-
amino Glutamate oxaloglutarate Lowering the activation energy. It is done by
transferase Pyruvate amino transferase creating an environment in which the transition
transaminase) state is stabilized (e.g. straining the shape of a
2.3 Gamma GGT 2.3.2.2 (5-Glutamyl )
substrateby binding the transition-state
Glutamyl peptide amino acid,
transferase 5- glutamyl conformation of the substrate/product molecules,
transferase the enzyme distorts the bound substrate(s) into their
2.4 Creatine CK 2.7.3.2 ATP-creatine, N- transition state form, thereby reducing the amount
kinase Phosphotransferase of energy required to complete the transition).
3. Alkaline ALP 3.1.3.1 Ortho-phosphoric,
Hydrolases Phosphatase monoester Providing an alternative pathway. This mechanism
phosphohydrolase can be illustrated for example, temporarily reacting
(alkaline optimum) with the substrate to form an intermediate Enzyme-
Acid ACP 3.1.3.2 Ortho-phosphoric, substrate (ES) complex, which would be impossible
Phosphatase monoester
phosphohydrolase
in the absence of the enzyme.
(acid optimum) Reducing the reaction entropy change. This can be
-Amylase AMS 3.2.1.1 1,4- D- Glucan, illustrated by bringing substrates together in the
Glucanohydrolase correct orientation to react. Considering enthalpy
4. Lyase Aldolase ALD 4.1.2.13 D Fructose
1,6 Bis phosphate ,
change (H) alone overlooks this effect. It is
D- glyceraldehyde, interesting to note that entropic effect involves
3-phosphate lyase destabilization of the ground state, and its
5. Triophosphate TPP 5.3.1.1 Triose phosphate contribution to catalysis is relatively small
Isomerase isomerase isomerase
6. Ligase Glutathione GSH-5 6.3.2.3 Gluthione
Synthetase Synthetase MODELS OF ENZYME ACTION
1) Lock and key hypothesis
1. Oxidoreductases 2) Induced Fit Hypothesis
The change in shape is 'induced' by the approaching
Catalyze redox reaction between two substrates
substrate molecule. This more sophisticated model
A- + B A + B-
relies on the fact that molecules are flexible because
E.g: Dehydrogenase (Lactate Dehydrogenase)
single covalent bonds are free to rotate.
2. Transferases
Catalyze the transfer of a group (Phosphate, methyl,
Enzyme Kinetics
etc.) between two substrates (A-X + B A + B-X)
E.g: Transferase (ALT, AST, GGT) and Kinase (CK) Catalytic Mechanism of Enzymes
3. Hydrolases o Enzymes catalyze physiologic reactions by
Catalyze hydrolysis of various bonds lowering the activation energy level that
AB + H2O AOH + BH the reactants must reach
E.g: Amylase (AMY), Lipase (LPS), Phosphatase (ALP, Relationship between Enzyme, Substrate and
ACP) Product
4. Lyases
Catalyze the removal of groups from substrates
without hydrolysis; the product remain double a) Absolute specificity
bonds Combines with only one substrate and catalyzes only
ATP cAMP + PPi one reaction (E.g. CK, LD)
E.g. Fructose biphosphate aldolase (ALS)
b) Group specificity
5. Isomerases
Combine with all substrates containing a particular
Catalyze the interconversion of geometric, optical or
positional isomers chemical group (E.g. ACP, ALP)
AB c) Bond specificity
E.g. Triphosphate isomerase (TPI) Specific to chemical bonds (E.g. AMY, LPS)
d) Sterioisometric specificity
Combine with one optical isomer (E.g. LDH, G6PD)
Order of Reaction
the order of the reaction can be specified in terms of iv. Temperature
the order with respect to each specific reactant or Within 0.1C
the overall order of the reaction. Enzyme is active at 25C, 30C, 37C.
Consider the reaction mA + nB<===> C. v. Cofactors
The rate equation is R = k[A]m[B]n. Activators: Metalic (Ca2+) and Non Metallic (Cl- )
If the exponent m in the equation is 1, then the Coenzymes (prosthetic groups): 2nd substrates (NAD)
reaction is said tobe First order with respect to A. vi. Inhibitors
If m = 2, In then, 2A + 1B <===> 1C) then it is said to An inhibitor is any compound that reduces the
be second order with respect to A and first order velocity of the enzyme-catalyzed reaction when
with respect to B. present in the reaction mixture.
Now, if m = 1 and n = 1, since it is first order with Penicillin irreversibly (covalently) inhibits an enzyme
respect to A and B, then the overall order of the involved in bacterial cell wall synthesis
reactionis said to be Second order (or m + n). Ibuprofen and many other nonsteroidal
HALF-LIFE antiinflammatory drugs (NSAIDs) are reversible
By definition, Half-life (t1/2) is the time required for competitive inhibitors of the cyclooxygenase activity
half of the original concentration of the limiting of prostaglandin H2 synthase.
reactant tobe used up as the reaction takes place or Inhibitors that occupy the active site and prevent a
half-life is equal to 0.69 /K. substrate molecule from binding to the enzyme are
Thus, the larger the rate constant (K), the faster will said to be active site-directed (or competitive, as
deplete the substrate. they 'compete' with the substrate for the active
As noted, in a first order reaction, the half-life is site).
inversely proportional to the rate constant (k). Inhibitors that attach to other parts of the enzyme
LOWERING ACTIVATION ENERGY molecule, perhaps distorting its shape, are said to be
1. Increase the proximity of the reactants, non-active site-directed (or non competitive)
2. Increase the concentration of the reactants, 1. Competitive Inhibition
3. Increase the surface area of the reactants In competitive inhibition, a chemical inhibitor
4. Increase the temperature of the reactants, competes for the active site with the substrates. The
5. Use a catalyst (a substance which speeds up a question immediately becomes:
chemical reaction but is not used up), Who gets to react with the active site - the inhibitor
6. Use an enyzme. or the substrate? The answer to this query depends
Factors that Influence Enzymatic Reactions upon the affinity of the enzyme for the substrate
i. Substrate Concentration and for the inhibitor. Often, the enzyme has a
First order kinetics (Michaelis- Menten hypothesis) greater affinity for the inhibitor than it does for the
o Reaction rate is proportional to the substrate.
substrate concentration. In case of Methanol poisoning, it occurs because
ii. Enzyme Concentration methanol is oxidized to formaldehyde and formic
Zero-order kinetics acid which attack the optic nerve causing blindness.
o Only a fixed number of substrate (in excess) Ethanol (an example of competitive inhibitor) is
is converted to product per second given as an antidote for methanol poisoning because
iii. pH ethanol competitively inhibits the oxidation of
Common range 7.0-8.0 methanol. It is shown when ethanol is oxidized in
Controlled by buffers preference to methanol.
Consequently, the oxidation of methanol is slowed
Enzymes Sources Substrates Optim down so that the toxic by-products do not have a
um pH
Sucrase Intestine Sucrose 6.2
chance to accumulate.
Ribonuclease Pancreas 3-50-Cytidylyl adenine 7.0 2. Uncompetitive Inhibition
- Glucosidase Yeast Methyl--D-glucoside 5.4 Occurs when the substrates fit into the active sites of
Acetylcholinesterase Erythrocytes Acetylcholine 7.5
the enzyme but an inhibitor prevents the release of
Enolase Rabbit 2-Phospho-D-Glycerate 6.8
Muscle the product or to stop enzyme from reacting with
Arginase Beef Liver L-Arginine 8.4-9.7 substrate to form the product
Pepsin Gastric Acetyl L-Phenylalanine , L- 1.5-2.5 It works well at higher substrate and enzyme
mucosa phenylalanine
concentrations that substrates are bonded to
enzymes.
 The formation of its binding site only forms when Enzymes of Clinical Significance
the enzyme and the substrate have interacted
amongst themselves.
It does not therefore work when additional
substrates are trying to be involved.
The enzyme-substrate-inhibitor complex does not
produce any product.
The binding results in decreasing concentration of
substrate binding to enzyme
3. Noncompetitive Inhibition
It is rare but there are instances in which it may be A. MI Profile
encountered.
It is a substance that interacts with the enzyme, but
usually not at the active site.
It reacts either remote from or very close to the
active site.
The net effect of a noncompetitive inhibitor is to
change the shape of the enzyme and thus the active
site, so that the substrate can no longer interact with
the enzyme to give a reaction.
One good example of noncompetitive inhibitor is the
nerve gases such diisopropylfluorophosphate (DFP)
that inhibits the active site of acetylcholine esterase
by reacting with the hydroxyl group of serine to
make an ester.

Measurement of Enzyme Activity

o Measurement of catalytic activity
in product concentration
in substrate concentration
or in coenzyme concentration (NADH) 1) Creatinine Kinase (CK)
in altered enzyme concentration Function, Tissue Source and Clinical Significance
Dependent on enzyme concentration Storage of high-energy creatine phosphate in
Performed in zero-order kinetics (linear phase) muscle cells
Highest activities in skeletal muscle, heart (AMI),
General methods of measuring enzymatic reaction and brain tissue
1. Fixed time (Two point) Assay
Reagents are combined and the amount of reaction
is measured (AMS, LPS, ACP, ALP)
2. Continuous-monitoring or kinetic assays Methods of Determination of Total CK
Measurements at specific time intervals a. Forward Reaction (Tanzer-Givarg)
Rate of change in substrate, cofactor, product. Measure in absorbance at 340 nm
Optimum pH is 9.0
Calculation of Enzyme Activity
IU (EC)
Amount of enzyme that will catalyze the reaction of
1 mol of substrate per minute (mol /min)
Kat (SI)
Amount of enzyme that will catalyze the reaction of
1 mol of substrate per second (mol/s)
b. Reverse Reaction (Oliver-Rosalki)
in absorbance at 340 nm
Measurement of Enzyme Mass
6x faster than forward reaction
Immunoassays and Electrophoresis
Optimum pH: 6.8
 3) Lactate Dehydrogenase (LDH)
Function, Tissue Source and Clinical Significance
Interconversion of lactate and pyruvate
Widely distributed, highest activities in heart,
hepatic, skeletal muscle and RBC
Diagnostic Significance of CK Isoenzyme
After MI, CK-MB (>6%) begin to rise within 4-8
hrs, peak at 12-24 hrs, and return to normal in
48-72 hrs.
Methods of Determination of CK Isoenzymes
Source of Error Methods of Determination of Total LDH
o Hemolysis cause false CK due to AK LD begin to rise within 10-24 hrs, peak at 48-72
activity hrs, and remains elevated for 10 days.
o CK is inactivated by light Reference Range: 100-225 U/L
o Physical activity and IM injections cause Wrobleuski Cabaud or Wacker method
CK Forward Reaction (Lactate Pyruvate)
Reference Range
in absorbance is monitored at 340 nm
o Male, 15-160 U/L : Female, 15-130 U/L
Optimal pH is 8.3 8.9
o CK-MB: <6% of total CK
Wrobleuski La Due
Reference Values:
Reverse Reaction (Pyruvate Lactate)
o 94-100% CK-MM
in absorbance is monitored at 340 nm
o 0-6% CK-MB
Optimal pH is 7.1 to 7.4
Other CK Isoenzymes
-hydroxybutyrate dehydrogenase (-HBD)
Macro-CK
Has greater affinity of H subunits
o Migrate midway CK-MM and CK-MB
Represent LDH-1
o CK-BB complexed with IgG/IgA
o CK-MM with LPP
Mitocondrial CK (CK-Mi)
o Migrates cathodal to CK-MM Diagnostic Significance of LDH Isoenzyme
o Bound to mitochondrial membranes Tetramer containing two active sub-units
2) Aspartate Aminotransferase (AST)
Function, Tissue Source and Clinical Significance
Serum glutamic-oxaloacetic transaminase
(SGOT)
Transfer of amino group in aspartate to -keto.
Involved in the synthesis and degradation of AA.
Methods of Determination of LDH Isoenzymes
Highest activities in cardiac, liver and skeletal
muscle. Relative concentration in normal serum:
o LDH-2>LDH-1>LDH-3>LDH-4>LDH-5
AST levels begin to rise in 6-8 hours, peak at 24
hours, and return to normal in 5 days. In AMI and Intravascular hemolysis, LDH-1 and
LDH-2 demonstrate a Flipped pattern
Also in hepatocellular and skeletal muscle dis.
(LDH-1 > LDH-2)

Methods of Determination of AST B. Liver Enzymes

Karmen Method 1. Alanine Aminotransferase (ALT)
o Uses malate dehydrogenase and monitors Function, Tissue Source and Clinical Significance
in absorbance at 340 nm Serum glutamic-pyruvic transaminase (SGPT)
o Falsely in hemolyzed sample Transfer of an amino group between alanine
o Reference Range: 5 30 U/L and -ketoglutarate
in hepatocellular disorders
 Methods of Determination of Total ALT Reference Range
Walker Method o 30 90 U/L (adult)
o Uses LD and monitors in absorbance o 70 220 U/L (0 3 months)
(340 nm) o 50 260 U/L (3 - 10 years)
o Reference Range: 6-37 U/L o 60 295 U/L (10 - puberty)
Diagnostic Significance of ALP Isoenzyme
Liver ALP
in liver diseases
Fractions: Major liver and fast liver (1) band
Bone ALP
Reitmann-Frankel in bone disease, healing of bone fractures
o Reagent: 2,4 dinitrophenyl hydrazine (2,4- and physiologic bone growth
DNPH) Placental ALP
o End Color: Brown in pregnancy
De Ritis Ratio Intestinal ALP
o The AST/ALT Ratio
Blood groups B or O, in fatty meal
o Differentiates the cause of hepatic
GIT disorders
disorder
Methods of Determination for ALP Isoenzymes
o Ratio > 1 Non viral origin (alcohol
Difference by Heat Stability
hepatitis)
o Serum is heated at 56C for 10 minutes
o Ratio < 1 Viral in origin
Liver ALP
o ALP residual activity is to >20%
Bone ALP
o ALP residual activity is to <20%
o Heat labile fraction
Selective Chemical Inhibition
o Placental and Intestinal ALP are inhibited by
phenylalanine (chemical inhibition)
Electrophoresis

2. Alkaline Phosphatase (ALP)

Function, Tissue Source and Clinical Significance
Catalyze the hydrolysis of phosphomonoesters
Requires Mg2+ activator
Evaluation of hepatobiliary and bone disorders.

3. Gamma-Glutamyltransferase (GGT)
Methods of Determination of ALP Function, Tissue Source and Clinical Significance
Bowers and McComb o Catalyze the transfer of the -glutamyl residue
o Based on molar absorptivity of p- from -glutamyl peptides to amino acids
Nitrophenol o Diagnosis hepatobiliary disorders (obstructive
liver disease) and chronic alcoholism

Methods of Determination for GGT

Szaz Assay
o Absorbance of p-Nitroaniline is measured at
405-420 nm
C. Pancreatic Enzymes D. Other Enzymes
1. Amylase (AMS) 1. Acid Phosphatase (ACP)
Function, Tissue Source and Clinical Significance Function, Tissue Source and Clinical Significance
Breakdown of starch via , 1-4 branching Catalyze the hydrolysis of phosphomonoesters
linkages Evaluation of metastatic carcinoma of prostate.
Increased in acute pancreatitis Forensic investigation of rape
Requires Ca2+ and Cl- for activation
Rise at 2-12 h, peak at 24 h and return to normal
within 3-5 d
Methods of Determinations
o Assay for Enzyme Activity
Methods of Determination of AMS o Reference Range: Prostatic ACP: 0 -3.5 ng/ml

Phosphatase inhibitors
a. L-tartrate ions
o inhibits specific prostatic ACP
o total ACP - ACP after inhibition =
prostatic ACP
b. Formaldehyde and Cupric ions
o inhibits red cell ACP

Amylase Isoenzymes
a) Salivary Amylase
ptyalin
fast moving
b) Pancreatic Amylase
amylopsin
slow moving
2. Lipase (LPS)
Function, Tissue Source and Clinical Significance
o Hydrolyzes of fats to produce alcohols and FA
o Earliest marker for acute pancreatitis
o Larger molecule, remains in circulation (7 days)

Methods of Determinations

PandaMT13