ENZYMES 2

Learning objectives and outcomes Overview of enzyme kinetics and factors affecting enzyme activity Overview of regulation of enzymes. Learning outcomes State the Michaelis-Menten equation and draw the Lineweaver-Burk plot. Briefly describe the effect of enzyme concentration, substrate concentration, temperature, pH and inhibitors on enzyme activity. Briefly describe the regulation of enzymes; allosteric regulation, covalent modification, proteolysis, induction and repression.

Enzyme kinetics Enzyme kinetics is the study of rates (velocities) of enzymatic reactions.

The Michaelis-Menten model accounts for the kinetic properties of many enzymes V = k2 [ES] The reaction rate is determined by how much of the enzyme is in a complex with substrate. Since [E] << [S], the maximum velocity occurs when all of the enzyme is in the ES complex 4

The Michaelis-Menten equation gives the reaction rate for any substrate concentration V max is a constant for a given enzyme Vmax is the theoretical maximal rate of the reaction 5

What does Km mean? Km (the Michaelis constant) is a measure of the affinity of the enzyme for the substrate. Substrates that bind tightly have small Km values, substrates that bind weakly have large Km values. 6

Example illustrating Km and affinity glucose + ATP  glucose 6 phosphate + ADP Glucokinase has High Km(10 mM) Hexokinase has Low Km (0.1 mM) Hexokinase has a low Km (high affinity) for glucose, so it permits initiation of glycolysis even when blood glucose levels are relatively low. Glucokinase requires a much higher glucose concentration for maximal activity. It is thus most active when glucose is very high in the portal vein, immediately after consumption of a carbohydrate-rich.

Lineweaver-Burk Plot 8

Factors Affecting the Actions of Enzymes Inhibitors Temperature pH Substrate concentration. Enzyme Concentration

Enzyme Inhibition Many different kinds of molecules inhibit enzymes and act in a variety of ways. Inhibition can be classified based on kinetics as Competetive Uncompetetive Non Competetive Inhibition can be Reversible Irreversible (Inhibitor is covalently bound, incapacitating the enzyme)

Competitive inhibition A competitive inhibitor is one which binds to the enzyme and prevents substrate binding at the active site; Competitive inhibitors are molecules with structures similar to the substrate called substrate analogs. They bind at the active site, but cannot be acted upon by the enzyme.

Competitive inhibitors alter the Km, not the Vmax

Example of competetive inhibition Both succinate and its structural analog malonate (–OOC—CH2—COO–) can bind to the active site of succinate dehydrogenase Malonate inhibits the enzyme from oxidizing succinate.

Non Competetive Inhibition

Noncompetitive inhibitors decrease the Vmax, but don’t affect the Km

Example of Non competetive inhibition Iodoacetate blocks the formation of 1,3-bisphosphoglycerate from glyceraldehyde-3-phosphate by inhibiting enzyme glyceraldehyde-3-phosphate dehydrogenase. Fluoride blocks the action of enolase, which converts 2-phosphoglycerate to phosphoenol pyruvate.

Uncompetetive inhibition Inhibitor dosen’t have affinity for free enzyme. In uncompetitive inhibition, inhibitor only binds ES and not E alone. Binds ES complex ↓ Vmax and Km Eg; Phenyl alanine inhibits placental alkaline phosphatase

Factors Affecting the Actions of Enzymes Inhibitors Enzyme Concentration Substrate concentration Temperature pH

Enzyme concentration The rate of enzyme catalyzed reaction is directly proportional to the concentration of enzyme. The plot of rate of catalysis versus enzyme concentrations a straight line

Michelis Menton Equation… Enzyme-catalyzed reactions exhibit saturation kinetics At low substrate concentration, there is a linear dependence between the rate and the substrate concentration. At high substrate concentration, the reaction is at the maximum velocity (Vmax)

Substrate concentration The rate of an enzyme-catalyzed reaction increases with substrate concentration until a maximal velocity (Vmax) is reached The leveling off of the reaction rate at high substrate concentrations reflects the saturation with substrate of all available binding sites on the enzyme molecules present. Most enzymes show Michaelis-Menten kinetics in which the plot of initial reaction velocity (vo) against substrate concentration ([S]), is hyperbolic In contrast, allosteric enzymes frequently show a sigmoidal curve

Effect of Temperature Enzyme activity increases with increase in temperature initially. After a critical temperature, the enzyme activity decreases with increase in the temperature.

Effect of pH The pH at which maximal enzyme activity is achieved is different for different enzymes, and often reflects the [H+] at which the enzyme functions in the body. For example, pepsin, a digestive enzyme in the stomach, is maximally active at pH 2, whereas other enzymes, designed to work at neutral pH, are denatured by such an acidic environment

REGULATION OF ENZYMES

FEEDBACK INHIBITION Inhibition of activity of enzyme of a biosynthetic pathway by the end product of that pathway is called as feedback inhibition. For example, formation of a substance D from A is catalyzed by three enzymes E1, E2 and E3. When enough D is formed it inhibit the activity of E1. By inhibiting E1, D regulates its own synthesis.

Example of Feedback inhibition Hexokinase is inhibited by the product of its reaction, glucose-6-phosphate. Inhibition of aspartate transcarbamoylase by CTP.

Allosteric Regulation Allosteric enzymes are enzymes that are regulated by molecules called effectors (also called modifiers) that bind noncovalently at a site other than the active site. These enzymes are usually composed of multiple subunits, and the regulatory site that binds the effector may be located on a subunit that is not itself catalytic. The presence of an allosteric effector can alter the affinity of the enzyme for its substrate, or modify the maximal catalytic activity of the enzyme, or both. Effectors that inhibit enzyme activity are termed negative effectors, whereas those that increase enzyme activity are called positive effectors.

Example of Allosteric regulation Aspartate transcarbamolyase It catalyzes first reaction unique to pyrimidine nucleotide biosynthesis. The enzyme consist of catalytic and regulatory subunits. It exists in less active form and high active form. Binding of CTP to regulatory subunit converts high active form to less active form. So CTP is called as negative effector or allosteric inhibitor. In contrast, binding of ATP to regulatory subunit convert less active form to high active form. So ATP is called as positive effector or allosteric activator.

Control of pyrimidine synthesis: regulation of aspartate transcarbamoylase activity Carbamoyl Phosphate + Aspartate --> N-carbamoyl Aspartate This is the first unique step in the synthesis of Pyrimidine Nucleotides and therefore the best point for regulation of this metabolic pathway Feedback Inhibition of ACT ase by products of the pathway 29

ATP and CTP alter the apparent Km of ATCase

Regulation by Covalent Modification This involves reversible covalent modification of the regulated enzyme Addition of a phosphoryl group to hydroxyl group of serine, threonine, or tyrosine residue Example Phosphorylation of glycogen phosphorylase converts less active to high active form. Dephosphorylation converts high active to less active form. Phosphorylation of glycogen synthase converts high active to less active form

Covalent modification regulates the catalytic activity of some enzymes

Regulation of Enzymes :Limited Proteolysis Enzymes are activated by cleavage of their polypeptide chain. Enzymes of this class are synthesized in inactive forms (proenzymes, or zymogens) and are activated by the proteolytic removal of short fragment from the amino terminus.

Regulation of digestive enzymes

Regulation of Enzymes : Control of Enzyme Production and Turnover The amount of an enzyme made by a cell may be regulated by increasing or decreasing the rate of either its synthesis or degradation Differs from the other method in that the total amount of enzyme polypeptide is changed without changing the catalytic properties of individual enzyme molecules Induced: amount of enzyme INCREASED Repressed: amount of enzyme DECREASED Example : Pyruvate carboxylase an enzyme of gluconeogenesis is induced by glucocorticoids and repressed by insulin.