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Enzymes

5.

Holoenzyme

Biologic proteins that catalyze biochemical reactions

Not consumed or changed in composition

Found in all body tissue (intracellular) and is ↑ in serum after cell injury

(intracellular) and is ↑ in serum after cell injury Function of Enzymes  Hydration of Carbon

Function of Enzymes

Hydration of Carbon Dioxide (respiration)

Nerve Induction

Muscle Contraction

Nutrient Degradation (Digestion)

Growth and Reproduction

Energy Storage and Use

Components of an Enzyme

1.

Active Site

A cavity of an enzyme where substrates bind and undergo a chemical reaction.

2.

Allosteric Site

A cavity other than the active site that binds regulatory (effector) molecules.

Terms associated with enzymes

1.

Substrates

 

Substances acted upon enzymes

Specific for each of their particular enzyme

2.

Cofactors

 

Non protein substances added in the enzyme substrate complex to manifest the enzyme activity

 

o

Coenzyme or Prosthetic group

 

An organic cofactor

Nucleotide (E.g. NAD, NADP) and Vitamins

 

o

Activator

 

An inorganic cofactor

Metal ion (E.g. Cl -1 , Mg ++ ,Cu + )

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

An active substance formed by combination of a co- enzyme and an apoenzyme.

6.

Proenzyme or Zymogens

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

Two kinds of allosteric enzymes:

1.

Homoallostery

This is a cooperative substrate binding and activation wherein substrate is a homotropic effector. Therefore the binding of substrate to one active site alters the substrate binding affinity and/or catalytic activity at other active sites on the multimeric enzyme.

2.

Heteroallostery

This merely involves the regulation by heterotropic effector molecules, which can be positive (activation) or negative (inhibition). These heterotropic effectors usually bind at a site other than the active site. Furthermore, these effectors can can activate or inhibit the activity of an enzyme.

Enzyme Classification and Nomenclature

The system for classification of enzymes that also serves as a basis for assigning code numbers to them.

These code numbers, prefixed by EC, which are now widely in use, contain four elements separated by points, with the following meaning as appearing in example: for Alcohol: NAD + oxidoreductase as EC number is 1.1.1.1

o

The first number shows to which of the six main divisions (classes) the enzyme belongs,

o

The second figure indicates the subclass,

o

The third figure gives the sub-subclass,

o

The fourth figure is the serial number of the enzyme in its sub-subclass.

CLASS

RECOMMENDE

ABBREVIATED

E.C

SCIENTIFIC NAME

D NAME

NAME

CODE

NO.

1.

Oxido

Lactate

LDH

1.1.1.27

L-Lactate NAD+

reductase

dehydrogenase

oxidoreductase

2.

2.1

Aspartate

SGOT ( Serum Glutamate Oxaloacetate transaminase)

2.6.1.1

L-Aspartate ,2-

Transferase

amino

oxaloglutarate

transferase

Amino transferase

 

2.2

Alanine

SGPT ( Serum Glutamate Pyruvate transaminase)

2.6.1.2

L-Alanine, 2-

amino

oxaloglutarate

transferase

amino transferase

 

2.3

Gamma

GGT

2.3.2.2

(5-Glutamyl ) peptide amino acid, 5- glutamyl transferase

Glutamyl

transferase

 

2.4 Creatine

CK

2.7.3.2

ATP-creatine, N-

kinase

Phosphotransferase

3.

Alkaline

ALP

3.1.3.1

Ortho-phosphoric,

Hydrolases

Phosphatase

monoester

phosphohydrolase

(alkaline optimum)

 

Acid

ACP

3.1.3.2

Ortho-phosphoric,

Phosphatase

monoester

phosphohydrolase

(acid optimum)

 

α-Amylase

AMS

3.2.1.1

1,4- D- Glucan, Glucanohydrolase

4.

Lyase

Aldolase

ALD

4.1.2.13

αD Fructose 1,6 Bis phosphate , D- glyceraldehyde, 3-phosphate lyase

5.

Triophosphate

TPP

5.3.1.1

Triose phosphate

Isomerase

isomerase

isomerase

6.

Ligase

Glutathione

GSH-5

6.3.2.3

Gluthione

 

Synthetase

Synthetase

1. Oxidoreductases

Catalyze redox reaction between two substrates

A - + B → A + B - E.g: Dehydrogenase (Lactate Dehydrogenase)

2. Transferases

Catalyze the transfer of a group (Phosphate, methyl, etc.) between two substrates (A-X + B → A + B-X) E.g: Transferase (ALT, AST, GGT) and Kinase (CK)

3. Hydrolases

Catalyze hydrolysis of various bonds

AB + H2O → A–OH + BH E.g: Amylase (AMY), Lipase (LPS), Phosphatase (ALP, ACP)

4. Lyases

Catalyze the removal of groups from substrates without hydrolysis; the product remain double bonds

ATP → cAMP + PPi E.g. Fructose biphosphate aldolase (ALS)

5. Isomerases

Catalyze the interconversion of geometric, optical or positional isomers

A → B E.g. Triphosphate isomerase (TPI)

6.

Ligases

Catalyze the joining of two substrate molecules, coupled with breaking of pyrophosphate bond in ATP

Ab + C → A–C + b E.g. Glutathione Synthetase (GSH-S)

ENZYME MECHANISMS

Lowering the activation energy. It is done by creating an environment in which the transition state is stabilized (e.g. straining the shape of a substrateby binding the transition-state conformation of the substrate/product molecules, the enzyme distorts the bound substrate(s) into their transition state form, thereby reducing the amount of energy required to complete the transition).

Providing an alternative pathway. This mechanism can be illustrated for example, temporarily reacting with the substrate to form an intermediate Enzyme- substrate (ES) complex, which would be impossible in the absence of the enzyme.

Reducing the reaction entropy change. This can be illustrated by bringing substrates together in the correct orientation to react. Considering enthalpy change (ΔH ) alone overlooks this effect. It is interesting to note that entropic effect involves destabilization of the ground state, and its contribution to catalysis is relatively small

MODELS OF ENZYME ACTION

1)

Lock and key hypothesis

2)

Induced Fit Hypothesis

The change in shape is 'induced' by the approaching substrate molecule. This more sophisticated model relies on the fact that molecules are flexible because single covalent bonds are free to rotate.

Enzyme Kinetics

Catalytic Mechanism of Enzymes

 
 

o

Enzymes catalyze physiologic reactions by lowering the activation energy level that the reactants must reach

Relationship between Enzyme, Substrate and Product

 
 

a)

Absolute specificity

Combines with only one substrate and catalyzes only one reaction (E.g. CK, LD)

b)

Group specificity

 

Combine with all substrates containing a particular chemical group (E.g. ACP, ALP)

c)

Bond specificity

 

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 the order with respect to each specific reactant or the overall order of the reaction.

Consider the reaction mA + nB<===> C.

The rate equation is R = k[A] m [B] n.

If the exponent m in the equation is 1, then the reaction is said tobe First order with respect to A.

If m = 2, In then, 2A + 1B <===> 1C) then it is said to be second order with respect to A and first order with respect to B.

Now, if m = 1 and n = 1, since it is first order with respect to A and B, then the overall order of the reactionis said to be Second order (or m + n). HALF-LIFE

By definition, Half-life (t1/2) is the time required for half of the original concentration of the limiting reactant tobe used up as the reaction takes place or half-life is equal to 0.69 /K.

Thus, the larger the rate constant (K), the faster will deplete the substrate.

As noted, in a first order reaction, the half-life is inversely proportional to the rate constant (k). LOWERING ACTIVATION ENERGY

1. Increase the proximity of the reactants,

2. Increase the concentration of the reactants,

3. Increase the surface area of the reactants

4. Increase the temperature of the reactants,

5. Use a catalyst (a substance which speeds up a

iv. Temperature

Within ±0.1°C

Enzyme is active at 25°C, 30°C, 37°C.

v. Cofactors

Activators: Metalic (Ca 2+ ) and Non Metallic (Cl - )

Coenzymes (prosthetic groups): 2 nd substrates (NAD)

vi. Inhibitors

An inhibitor is any compound that reduces the velocity of the enzyme-catalyzed reaction when present in the reaction mixture.

Penicillin irreversibly (covalently) inhibits an enzyme involved in bacterial cell wall synthesis

Ibuprofen and many other nonsteroidal antiinflammatory drugs (NSAIDs) are reversible competitive inhibitors of the cyclooxygenase activity of prostaglandin H2 synthase.

Inhibitors that occupy the active site and prevent a substrate molecule from binding to the enzyme are said to be active site-directed (or competitive, as they 'compete' with the substrate for the active site).

Inhibitors that attach to other parts of the enzyme molecule, perhaps distorting its shape, are said to be non-active site-directed (or non competitive)

1. Competitive Inhibition

In competitive inhibition, a chemical inhibitor competes for the active site with the substrates. The question immediately becomes:

 

chemical reaction but is not used up),

Who gets to react with the active site - the inhibitor

In case of Methanol poisoning, it occurs because

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 concentration.

substrate.

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) is converted to product per second

Ethanol (an example of competitive inhibitor) is 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.

Enzymes

Sources

Substrates

Optim

um pH

Sucrase

Intestine

Sucrose

6.2

Ribonuclease

Pancreas

3’-5’0-Cytidylyl adenine

7.0

Α- Glucosidase

Yeast

Methyl-α-D-glucoside

5.4

Acetylcholinesterase

Erythrocytes

Acetylcholine

7.5

Enolase

Rabbit

2-Phospho-D-Glycerate

6.8

Muscle

Arginase

Beef Liver

L-Arginine

8.4-9.7

Pepsin

Gastric

Acetyl L-Phenylalanine , L- phenylalanine

1.5-2.5

mucosa

Consequently, the oxidation of methanol is slowed down so that the toxic by-products do not have a chance to accumulate. 2. Uncompetitive Inhibition Occurs when the substrates fit into the active sites of the enzyme but an inhibitor prevents the release of the product or to stop enzyme from reacting with substrate to form the product

It works well at higher substrate and enzyme concentrations that substrates are bonded to enzymes.

The formation of its binding site only forms when 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 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)

in altered enzyme concentration

Dependent on enzyme concentration

Performed in zero-order kinetics (linear phase)

General methods of measuring enzymatic reaction 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

Measurements at specific time intervals

Rate of change in substrate, cofactor, product.

Calculation of Enzyme Activity

IU (EC)

Amount of enzyme that will catalyze the reaction of

1 μmol of substrate per minute (μmol /min)

Kat (SI)

Amount of enzyme that will catalyze the reaction of

1 mol of substrate per second (mol/s)

Measurement of Enzyme Mass

Immunoassays and Electrophoresis

Enzymes of Clinical Significance

and Electrophoresis Enzymes of Clinical Significance A. MI Profile 1) Creatinine Kinase (CK)  Function,

A. MI Profile

Enzymes of Clinical Significance A. MI Profile 1) Creatinine Kinase (CK)  Function, Tissue Source and
Enzymes of Clinical Significance A. MI Profile 1) Creatinine Kinase (CK)  Function, Tissue Source and

1)

Creatinine Kinase (CK)

Function, Tissue Source and Clinical Significance

Storage of high-energy creatine phosphate in muscle cells

Highest activities in skeletal muscle, heart (AMI), and brain tissue

activities in skeletal muscle, heart (AMI), and brain tissue  Methods of Determination of Total CK

Methods of Determination of Total CK

a. Forward Reaction (Tanzer-Givarg)

Measure in absorbance at 340 nm

Optimum pH is 9.0

↓ in absorbance at 340 nm  Optimum pH is 9.0 b. Reverse Reaction (Oliver-Rosalki) 

b. Reverse Reaction (Oliver-Rosalki)

in absorbance at 340 nm

6x faster than forward reaction

Optimum pH: 6.8

 Diagnostic Significance of CK Isoenzyme  After MI, CK-MB (>6%) begin to rise within

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

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

activities in heart, hepatic, skeletal muscle and RBC  Source of Error  Methods of Determination

Source of Error

Methods of Determination of Total LDH

o

Hemolysis cause false ↑ CK due to AK activity

o

CK is inactivated by light

o

Physical activity and IM injections cause ↑ CK

Reference Range

o

Male, 15-160 U/L : Female, 15-130 U/L

o

CK-MB: <6% of total CK

Reference Values:

o

94-100% CK-MM

o

0-6%

CK-MB

Other CK Isoenzymes

Macro-CK

o

Migrate midway CK-MM and CK-MB

o

CK-BB complexed with IgG/IgA

o

CK-MM with LPP

Mitocondrial CK (CK-Mi)

LD begin to rise within 10-24 hrs, peak at 48-72 hrs, and remains elevated for 10 days.

Reference Range: 100-225 U/L

Wrobleuski Cabaud or Wacker method

Forward Reaction (Lactate Pyruvate)

↑ in absorbance is monitored at 340 nm

Optimal pH is 8.3 8.9

Wrobleuski La Due

Reverse Reaction (Pyruvate Lactate)

↓ in absorbance is monitored at 340 nm

Optimal pH is 7.1 to 7.4

α-hydroxybutyrate dehydrogenase (α-HBD)

Has greater affinity of H subunits

Represent LDH-1

 Has greater affinity of H subunits  Represent LDH-1 o Migrates cathodal to CK-MM 

o

Migrates cathodal to CK-MM

o

Bound to mitochondrial membranes

Diagnostic Significance of LDH Isoenzyme

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 α-keto.

Involved in the synthesis and degradation of AA.

Highest activities in cardiac, liver and skeletal muscle.

AST levels begin to rise in 6-8 hours, peak at 24 hours, and return to normal in 5 days.

Also ↑ in hepatocellular and skeletal muscle dis.


 Also ↑ in hepatocellular and skeletal muscle dis.   Methods of Determination of LDH

Methods of Determination of LDH Isoenzymes

Relative concentration in normal serum:

o

LDH-2>LDH-1>LDH-3>LDH-4>LDH-5

In AMI and Intravascular hemolysis, LDH-1 and LDH-2 demonstrate a Flipped pattern (LDH-1 > LDH-2)

Methods of Determination of AST

Karmen Method

B. Liver Enzymes

1. Alanine Aminotransferase (ALT)

o

Uses malate dehydrogenase and monitors ↓ in absorbance at 340 nm

o

Falsely ↑ in hemolyzed sample

o

Reference Range: 5 30 U/L

Function, Tissue Source and Clinical Significance

Serum glutamic-pyruvic transaminase (SGPT)

Transfer of an amino group between alanine and α-ketoglutarate

↑ in hepatocellular disorders

(SGPT)  Transfer of an amino group between alanine and α -ketoglutarate  ↑ in hepatocellular
(SGPT)  Transfer of an amino group between alanine and α -ketoglutarate  ↑ in hepatocellular

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)

Range: 6-37 U/L o 60 – 295 U/L (10 - puberty)  Reitmann-Frankel o Reagent: 2,4

Reitmann-Frankel

o

Reagent: 2,4 dinitrophenyl hydrazine (2,4- DNPH)

o

End Color: Brown

De Ritis Ratio

o

The AST/ALT Ratio

o

Differentiates the cause of hepatic disorder

o

Ratio > 1 Non viral origin (alcohol hepatitis)

o

Ratio < 1 Viral in origin

hepatitis) o Ratio < 1  Viral in origin 2. Alkaline Phosphatase (ALP)  Function, Tissue

2. Alkaline Phosphatase (ALP)

Function, Tissue Source and Clinical Significance

Catalyze the hydrolysis of phosphomonoesters

activator

Evaluation of hepatobiliary and bone disorders.

Requires Mg2+

of hepatobiliary and bone disorders. Requires Mg2+  Methods of Determination of ALP  Bowers and

Methods of Determination of ALP

Bowers and McComb

o Based on molar absorptivity of p- Nitrophenol

McComb o Based on molar absorptivity of p - Nitrophenol  Diagnostic Significance of ALP Isoenzyme
McComb o Based on molar absorptivity of p - Nitrophenol  Diagnostic Significance of ALP Isoenzyme

Diagnostic Significance of ALP Isoenzyme

Liver ALP

↑ in liver diseases

Fractions: Major liver and fast liver (α1) band

Bone ALP

↑ in bone disease, healing of bone fractures and physiologic bone growth

Placental ALP

↑ in pregnancy

Intestinal ALP

Blood groups B or O, ↑ in fatty meal

↑ GIT disorders

Methods of Determination for ALP Isoenzymes

Difference by Heat Stability

o Serum is heated at 56°C for 10 minutes 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

by phenylalanine (chemical inhibition)  Electrophoresis 3. Gamma-Glutamyltransferase (GGT)  Function, Tissue

3. Gamma-Glutamyltransferase (GGT)

Function, Tissue Source and Clinical Significance

o

Catalyze the transfer of the γ-glutamyl residue from γ-glutamyl peptides to amino acids

o

Diagnosis hepatobiliary disorders (obstructive liver disease) and chronic alcoholism

disorders (obstructive liver disease) and chronic alcoholism  Methods of Determination for GGT  Szaz Assay

Methods of Determination for GGT

Szaz Assay

o

Absorbance of p-Nitroaniline is measured at 405-420 nm

 Methods of Determination for GGT  Szaz Assay o Absorbance of p-Nitroaniline is measured at

C. Pancreatic Enzymes

1. Amylase (AMS)

Function, Tissue Source and Clinical Significance

Breakdown of starch via α, 1-4 branching linkages

Increased in acute pancreatitis

Requires Ca 2+ and Cl - for activation

Rise at 2-12 h, peak at 24 h and return to normal within 3-5 d

D. Other Enzymes

1. Acid Phosphatase (ACP)

Function, Tissue Source and Clinical Significance

Catalyze the hydrolysis of phosphomonoesters

Evaluation of metastatic carcinoma of prostate.

Forensic investigation of rape

carcinoma of prostate.  Forensic investigation of rape  Methods of Determinations o Assay for Enzyme
 Methods of Determinations

Methods of Determinations

o

Assay for Enzyme Activity

Methods of Determination of AMS

o

Reference Range: Prostatic ACP: 0 -3.5 ng/ml

of AMS o Reference Range: Prostatic ACP: 0 -3.5 ng/ml  Amylase Isoenzymes a) Salivary Amylase
of AMS o Reference Range: Prostatic ACP: 0 -3.5 ng/ml  Amylase Isoenzymes a) Salivary Amylase
of AMS o Reference Range: Prostatic ACP: 0 -3.5 ng/ml  Amylase Isoenzymes a) Salivary Amylase

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)

o Larger molecule, remains in circulation (7 days)  Methods of Determinations  Phosphatase inhibitors

Methods of Determinations

in circulation (7 days)  Methods of Determinations  Phosphatase inhibitors a. L-tartrate ions o inhibits
in circulation (7 days)  Methods of Determinations  Phosphatase inhibitors a. L-tartrate ions o inhibits

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

o total ACP - ACP after inhibition = prostatic ACP b. Formaldehyde and Cupric ions o

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