Isoenzymes. Enzymodiagnostics. Enzymopathy. Enzymotherapy

Definition Enzymes are protein catalysts for biochemical reactions in living cells They are among the most remarkable biomolecules known because of their extraordinary specificity and catalytic power, which are far greater than those of man-made catalysts.

Isoenzymes These are the enzymes from the same organism which catalyse the same reaction but are chemically and physically distinct from each other.

Lactate dehydrogenase It occurs in 5 possible forms in the blood serum: LDH1 LDH2 LDH3 LDH4 LDH5

Structure of LDH Each contains 4 polypeptide chains which are of 2 types: A and B which are usually called M (muscle) and H (heart). LDH1 –H H H H LDH2 – H H H M LDH3 – H H M M LDH4 – H M M M LDH5 – M M M M

Clinical importance of LDH Acute myocardial infarction LDH1 and LDH2 Acute liver damage LDH4 and LDH5

Creatine kinase It has 3 isoenzymes: CK1 CK2 CK3 Clinical importance: When patient have acute myocardial infarction CK appears in the blood 4 to 8 hours after onset of infarction and reaches a peak in activity after 24 hours.

Enzyme-Activity Units The most widely used unit of enzyme activity is international unit defined as that amount which causes transformation of 1.0 mkmol of substrate per minute at 25°C under The specific activity is the number of enzyme units per milligram of protein.

Enzyme-Activity Units The molar or molecular activity, is the number of substrate molecules transformed per minute by a single enzyme molecule The katal (abbreviated kat), defined as the amount of enzyme that transforms 1 mol of substrate per 1 sec.

Naming The name enzyme (from Greek word "in yeast") was not used until 1877, but much earlier it was suspected that biological catalysts are involved in the fermentation of sugar to form alcohol (hence the earlier name "ferments"). ").

Naming and Classification of Enzymes Many enzymes have been named by adding the suffix -ase to the name of the substrate, i.e., the molecule on which the enzyme exerts catalytic action. For example, urease catalyzes hydrolysis of urea to ammonia and CO2, arginase catalyzes the hydrolysis of arginine to ornithine and urea, and phosphatase the hydrolysis of phosphate esters.

Classification of enzymes Oxido-reductases (oxidation-reduction reaction). Transferases (transfer of functional groups). Hydrolases (hydrolysis reaction). Lyases (addition to double bonds). Isomerases (izomerization reactions). Ligases (formation of bonds with ATP cleavage).

The structure of enzymes Protein part + Non- protein part Apoenzyme + Cofactor = Holoenzyme Function of apoenzyme: It is responsible for the reaction Function of cofactor: It is responsible for the bonds formation between enzyme and substrate Transfer of functional groups Takes plase in the formation of tertiary structure of protein part

Cofactor 1. Prosthetic group (when cofactor is very tightly bound to the apoenzyme and has small size ) 2. Metal ion 3. Coenzyme(organic molecule derived from the B vitamin which participate directly in enzymatic reactions)

Prosthetic group 1. Heme group of cytochromes 2. Biothin group of acetyl-CoA carboxylase

Metal ions Fe - cytochrome oxidase, catalase Cu - cytochrome oxidase, catalase Zn - alcohol dehydrogenase Mg - hexokinase, glucose-6-phosphatase K, Mg - pyruvate kinase Na, K – ATP-ase

Coenzyme B1 TPP- Thiamine Pyro Phosphate B2 FAD- Flavin Adenine Dinucleotide FMN- Flavin Mono Nucleotide Pantothenic acid Coenzyme A (CoA) B5 NAD – Nicotinamide Adenine Dinucleotide NADP- Nicotinamide Adenine Dinucleotide Phosphate

Chemical Kinetics

The Michaelis-Menten Equation In 1913 a general theory of enzyme action and kinetics was developed by Leonor Michaelis and Maud Menten. 1. Point А. 2. Point В. 3. Point С.

Mechanism of enzyme reaction 1. Formation of enzyme – substrate complex E + S → ES 2. Conversion of the substrate to the product ES→ EP 3. Release of the product from the enzyme EP → E+P

The Free Energy of Activation Before a chemical reaction can take place, the reactants must become activated. This needs a certain amount of energy which is termed the energy of activation. It is defined as the minimum amount of energy which is required of a molecule to take part in a reaction.

The Free Energy of Activation For example,decomposition of hydrogen peroxide without a catalyst has an energy activation about 18 000. When the enzyme catalase is added, it is less than 2000.

The Free Energy of Activation The rate of the reaction is proportional to the energy of activation: Greater the energy of activation Slower will be the reaction While if the energy of activation is less, The reaction will be faster

Energy of Activation

Effect of pH on Enzymatic Activity Most enzymes have a characteristic pH at which their activity is maximal (pH- optimum); above or below this pH the activity declines. Although the pH-activity profiles of many enzymes are bell-shaped, they may be very considerably in form.

Effect of pH on Enzymatic Activity

Effect of Temperature on Enzymatic Reactions .The rate of enzyme catalysed reaction generally increases with temperature range in which the enzyme is stable. The rate of most enzymatic reactions doubles for each 100 C rise in temperature. This is true only up to about 500 C. Above this temperature, we observe heat inactivation of enzymes. The optimum temperature of an enzyme is that temperature at which the greatest amount of substrate is changed in unit time.

Effect of Temperature on Enzymatic Reactions

Allosteric enzymes Allosteric enzymes have a second regulatory site (allosteric site) distinct from the active site Allosteric enzymes contain more than one polypeptide chain (have quaternary structure). Allosteric modulators bind noncovalently to allosteric site and regulate enzyme activity via conformational changes

2 types of modulators (inhibitors or activators) • Negative modulator (inhibitor) –binds to the allosteric site and inhibits the action of the enzyme –usually it is the end product of a biosynthetic pathway - end-product (feedback) inhibition • Positive modulator (activator) –binds to the allosteric site and stimulates activity –usually it is the substrate of the reaction

Example of allosteric enzyme - phosphofructokinase-1 (PFK-1) PFK-1 catalyzes an early step in glycolysis Phosphoenol pyruvate (PEP), an intermediate near the end of the pathway is an allosteric inhibitor of PFK-1 PEP

Regulation of enzyme activity by covalent modification Covalent attachment of a molecule to an amino acid side chain of a protein can modify activity of enzyme

Phosphorylation reaction

Dephosphorylation reaction Usually phosphorylated enzymes are active, but there are exceptions (glycogen synthase) Enzymes taking part in phospho-rylation are called protein kinases Enzymes taking part in dephosphorylation are called phosphatases

Examples of specific proteolysis Activation by proteolytic cleavage • Many enzymes are synthesized as inactive precursors (zymogens) that are activated by proteolytic cleavage • Proteolytic activation only occurs once in the life of an enzyme molecule Examples of specific proteolysis •Digestive enzymes –Synthesized as zymogens in stomach and pancreas •Blood clotting enzymes –Cascade of proteolytic activations •Protein hormones –Proinsulin to insulin by removal of a peptide

Multienzyme Complexes and Multifunctional Enzymes Multienzyme complexes - different enzymes that catalyze sequential reactions in the same pathway are bound together Multifunctional enzymes - different activities may be found on a single, multifunctional polypeptide chain

Metabolite channeling Metabolite channeling - “channeling” of reactants between active sites Occurs when the product of one reaction is transferred directly to the next active site without entering the bulk solvent Can greatly increase rate of a reactions Channeling is possible in multienzyme complexes and multifunctional enzymes

Enzyme Inhibition Reversible inhibition A. Competitive B. Non-competitive C. Uncompetitive 2. Irreversible inhibition

Competitive Inhibition

Usage competitive inhibition in medicine The antibacterial effects of sulfanilamides are also explained by their close resemblance to para-amino-benzoic acid which is a part of folic acid, an essential normal constituent of bacterial cells. The sulfanilamides inhibit the formation of folic acid by bacterial cells and thus the bacterial multiplication is prevented and they soon die.

Non-competitive Inhibition In this case, there is no structural resemblance between the inhibitor and the substrate. The inhibitor does not combine with the enzyme at its active site but combines at some other site. E + S +I =ESI (INACTIVE COMPLEX) E + S = ES ES + I = ESI

Uncompetitive inhibition E + S +I =ESI (No active complex)

Irreversible Inhibition The inhibitor is covalently linked to the enzyme. The example: Action of nerve gas poisons on acetylcholinesterase,an enzyme that has an important role in the transmission of nerve impulse.