CLINICAL ENZYMOLOGY: PROPERTIES OF ENZYMES

 

Introduction


Enzymes are essential to physiologic functions, such as…

n      Hydration of CO2

n      Nerve conduction

n      Muscle contraction

n      Nutrient degradation

n      Energy utilization

 

Definition of an Enzyme:

Enzymes are proteins in nature and are synthesized in the body in the same manner as all other proteins.  Its role is that of biochemical catalyst.  Each biochemical reaction which occurs in the body has a specified enzyme associated with it.

 

The Generic Enzyme Reaction:

E + S ß→ ES → E + P

E = enzyme
S = substrate (the material acted on by the enzyme)
P = product of the reaction

 

Formation of the enzyme/substrate complex is reversible; the complex can either proceed onward to product or can dissociate back to separate enzyme and substrate molecules, with no reaction taking place.


Enzymes and Metabolic Pathways


 E1                   E2           E3                 E4
A         à        B  à       C     à        D  à      E…
                                                            or D à F

 

Factors Affecting Reaction Rate

 

n      Substrate Concentration

n      Enzyme Concentration

n      Effect of pH on Rate

n      Temperature

n      Coenzyme Concentration

n      Inhibitor Concentration

n      Activator Concentration

n      Buffer

 

 

Substrate Concentration
- as the substrate level increases, the enzyme reaction rate increases
- there comes a point where a further increase in substrate concentration produces no more enhancement of the reaction rate

Michaelis-Menten hypothesis:
The rate of conversion of substrate to product in a reaction is determined by the substrate conc. & the rate of dissociation of enzyme-substrate complex.

 

Enzyme Concentration


Effect of pH on Rate
 - at the pH extremes, whatever they may be, the reaction rate is rather low
 - the rate gradually increases to a pH optimum, the point at which the reaction rate is greatest for the conditions
 - further extremes of pH may cause hydrolysis of portions of the enzyme, leading to loss of structural integrity & decrease in activity

 

Temperature
- can increase the rate of an enzyme reaction
 - enzyme activity doubles (more or less) for every 10oC rise in temperature
- after a certain point, further increase in temp. Leads not to further increase in rate, but to loss of enzyme activity
- freezing does not affect most enzymes
-inactivation occurs at 60-65oC (denaturation of proteins)

 

Coenzyme Concentration
- most commonly used coenzyme in clinical settings is NAD (or NADH)
- not tightly bound to the enzyme but exists free
- hasten enzymatic reaction; but they undergo a change or is consumed to another product; serve as second substrates
- pyridoxal phosphate (a form of VitB6) is important in aminotransferase reaction

 

Inhibitor Concentration
- inhibitors (molecules which decrease the rate of an enzyme reaction)
-may bind to the active site, blocking the access of substrate to the enzyme
a.) Competitive inhibitor – competes with a normal enzyme-substrate by binding to the same site on an enzyme
b.) Non-competitive inhibitor – binds to the other site on the enzyme

 

Activator Concentration
- these inorganic entities help bind the substrate to the active site by forming ionic bridges (altering spatial configuration of the enzyme for proper substrate binding, linking substrate to the enzyme or a coenzyme)
- usually the activator is a cation generally a +2 species (ex. Zn2+;Mg2+)
- the only known anion activator is Cl-

 

Buffer
-play an important role not only in the regulation of pH
-best known buffer contribution to enzyme reaction is in the alkaline phosphatase system (the buffer serves as an acceptor of the phosphate group removed from the substrate; without this acceptor, the rate of the reaction is markedly lower)

 

 

Enzyme Nomenclature
- designated according to the reaction it catalyzed.  Some characteristic of the reaction was identified (usually the substrate) and the suffix –ase was added to the name.
-ex. Lipids (lipid + ase) > lipase
disaccharides > disaccharidases

 

There are over 1500 different enzymes… so it became necessary for the International Union of Biochemistry (IUB, affiliated with IUPAC), to establish nomenclature rules for enzymes.
There are six categories based on the type of reaction

 

ENZYME CLASSES

 

Class

Category

Type of Reaction Catalyzed

1

2

 

3

4

 

 

5

6

Oxidoreductase

Transferase

 

Hydrolase

Lyases

 

 

Isomerases

Ligases

Oxidation/Reduction Reactions

Transfer of intact group of atoms from one molecule to another

Cleavage of bonds w/ water

Cleavage of C-C, C-O, C-N or other types of bonds; does not involve water

Convert one isomer to another

Bond formation between two groups of atoms; with ATP as energy source

 

 

EC Class 1: Oxidoreductase
- the addition of hydrogen to a double bond (reduction) or the removal of hydrogen from a molecule to leave a double bond (oxidation)
- older name: dehydrogenase
- ex. lactate dehydrogenase
(impt info in cases of heart attacks/liver problems)
pyruvate + NADH + H+ à lactate + NAD
- ex. alcohol dehydrogenase (conv. of ethanol to acetaldehyde in the liver)

 

 

EC Class 2: Transferases
- move an intact group of atoms from one molecule to another
- the group moved is a functional group, such as an amine or a phosphate entity)
- ex. creatine kinase
    ATP+creatine à ADP + creatine phosphate
- transaminase process
amino acid I+keto acid II à keto acid I+amino acid II

 

EC Class 3: Hydrolases
- enzymes involved in the splitting of molecules, with water as part of the rxn process
- amylase with the cleavage of
–C—O—C— bonds in starch
- lipase breaks down triglycerides to form glycerol and free fatty acids
-acid and alkaline phosphatases

 

EC Class 4: Lyases
- split molecules (lysis means “splitting”)
- bonds broken may be C—C; C—O; C—N, or other bonds
- aldolase (cleaves the 6-carbon molecule fructose-1,6-diphosphate to produce two 3-carbon compounds: glyceraldehyde-3-phosphate and dihydroxyacetone phosphate

 

EC Class 5: Isomerases
- conversion of one isomer to another
- transformations may include change of cis to trans, of an L-form of a compound to the corresponding D-form, or an aldehyde to a ketone
- generally reversible

 

EC Class 6:  Ligases
- cause bond formation between two molecules to form a larger molecule
- a requirement is the breakdown of ATP, which provides the biochemical energy necessary for the reaction to take place
- ex. aminoacyl-tRNA synthetases
ATP + L-tyrosine + tRNA à AMP
+ pyrophosphate + L-tyrosyl-tRNA

 

MEASUREMENT OF ENZYME ACTIVITY:
*For clinical diagnostic situation
*Enzyme changes in Pathological Conditions
> the changes in circulating enzyme level is often a reflection of damage to a particular tissue à a marker of cellular destruction

 

Enzyme level and tissue specificity
> each enzyme has defined tissue specificity
> by knowing the tissue specificity of any enzyme, we can better ascertain where the damage occurred in the body

 

 

 

 

Tissue Specificity of Enzymes

 

 

Enzyme

Principal Tissue(s)

High Specificity

Acid phosphatase

Alanine aminotransferase

Amylase

Lipase

Erythrocytes, prostate

Liver

Pancreas, salivary glands

Pancreas

Moderate specificity

Aspartate aminotransferase

Creatine kinase

Liver, heart, skeletal muscle

 

Heart, skeletal muscle, brain

Low specificity

Alkaline phosphatase

Lactate dehydrogenase

Liver, bone, kidney

All tissues

 

 

Substrate Disappearance or Product Formation
- measure the change in concentration as a function of time
- the material quantitated can be either substrate or product

 

Types of Enzyme Assays:
1. END-POINT ANALYSIS
-the simplest and most widely used technique
-the reaction is initiated by the addition of substrate and allowed to proceed for a set period of time
-at the end of that time, the reaction is stopped and the amount of product formed during the time is measured
-problem: substrate depletion

 

2. MULTIPOINT ASSAY
- measures the change in concentration at several intervals during the course of the assay
-practical only with automated analysis systems for laboratories

 

3. KINETIC ASSAY
- involves the continuous measurement of change in concentration as a function of time
-places the reaction cuvet in a spectro, sets the instrument at the desired wavelength, and runs a strip chart to trace the change in absorbance as a line on the chart
-has a value in a research setting

 

 

 

 

4. COUPLED-ENZYME REACTIONS
- assay the product of one enzyme reaction with the use of another enzyme reaction
-ex. aminotransferase activity

Reaction One:                      aspartate aminotransferase
alpha-ketoglutarate+aspartate à glutamate+oxaloacetate

Reaction Two:                 malate dehydrogenase                             
          oxaloacetate + NADH à malate + NAD

 

 

 

 

Enzyme Measurement
I.U. or U/L – amount of substrate utilized, or product produced, in terms of μmol/minute per liter of blood and other body fluids under controlled conditions
katal – more recently has been recommended as a unit of enz.act.
  - amount of enz w/c converts 1 mol of substrate per second

 

Effect of Some Interfering Substances in Enzyme Reaction:
1. Hemolysis – causes falsely elevated value due to release of some enzymes by red blood cells
2. Lactescence or milky serum – causes variable absorption by the spectrophotometer

3. Storage – best at -20oC for longer period of time
- for substrate and co-enzyme use, ref temp (2-8oC)
- for LD1 and LD5, room temp only coz it is inactivated at ref temp
-CK should be stored at -70oC

4. Accuracy in timing
5. Meticulously clean glassware
6. pH

   - ex. LD
pyruvate to lactate (pH 7 to 8)
lactate to pyruvate (pH 9 to10)