Principles of Biochemistry: Enzymes

Enzymes Enzyme Structure • Enzymes are biological catalysts • Most enzymes are proteins • They are not consumed or altered during the reaction • Enzymes may require a non-peptide component as a cofactor. The peptide component is called the apoenzyme, the cofactor is called as the coenzyme and the combined functional unit is the holoenzyme. • They do not change the equilibrium, just reduce the time required to reach equilibrium. • They increase the rate of a reaction by lowering the activation energy barrier. • The reactant usually binds to the enzyme forming a transition complex that stabilizes the transition state. • The interaction between enzyme and substrate is very specific. Specificity may be for a class of compounds or for a particular compound. Eg. Hexokinase / glucokinase Enzyme Classification • Every enzyme is identified by a unique set of 4 numbers. • There are 6 main classes based on the type of reaction catalyzed. This makes up the first number of the enzyme identity. • Oxidoreductase: oxidation-reduction reaction. (Dehydrogenases) • Transferase: transfer of intact functional group • Hydrolase: hydrolysis (addition or removal of water) • Lyase: Cleavage of a C-C or C-N or C-O bond. Removal (or addition) of water across double bond. • Isomerase: Isomerization (LÙD, cisÙtrans, CHOÙC=O etc.) • Ligase: Joining of two molecules requiring energy from ATP. • Cofactors that are tightly bound to the polypeptide are called prosthetic groups. Such proteins are called as complex or conjugated proteins. Proteins without prostetic groups are simple proteins • Common coenzymes are vitamins and metal ions Reaction Rate and Enzyme Activity • An enzyme increases the rate of a reaction. Reaction rate is defined as the amount of reactant modified per unit time. Units for reaction rate are µmoles/sec or mol/min. • Enzymes are required in minute quantities and enhance reaction rates by 1010- to 1020-fold • Enzyme activity is the ability of an enzyme to modify a reactant. 1 unit (U) is the enzyme activity that converts 1 µmole of reactant per min under standard conditions. • The specific activity of an enzyme is defined as the activity per unit of mass or U/mg protein 1 Substrate Binding and Enzyme Action • The first step in a enzyme catalyzed reaction is the formation of the enzyme-substrate complex. This is represented by the equation: E + S = ES = E + P. • The region of the enzyme where the substrate binds is called as the active site. This consists of a substrate binding site and the catalytic site. • The active site is usually a cleft or pocket created by the unique tertiary structure of the enzyme protein • Enzyme specificity is due to specificity of substrate binding driven by substrate and enzyme 3D structure • The ES complex is stabilized in the transition state by non-covalent interactions between substrate the the aa in the active site. Factors Affecting Reaction Rates • The study of enzyme reaction rates is called enzyme kinetics. Enzyme kinetics are affected by: • Temp and pH: Each enzymatic reaction has an optimum pH and optimum temperature. Extreme temp or pH disrupts enzyme structure and therefore reaction rate • Enzyme concentration: At saturating substrate concentration, the initial velocity is directly related to the enzyme concentration. E + S = ES = E + P. Thus, as long as S is not limiting, more E leads to more ES. • Substrate concentration: The reaction rate = k [P] / [S]. The rate can be increased by adding more substrate, or by removing product as it is formed Models of Enzyme-Substrate Binding • Lock and key model. This means that the enzyme’s tertiary structure consists of a unique pocket or site which is tailor-made to fit only its substrate and nothing else, just as a key fits into a lock. • Induced-fit model: This updated model states that enzymes interact with substrates and in the process change their conformation such that the enzyme is snug around the substrate, sort of like a glove around a hand. Michaelis-Menten Equation for Enzyme Kinetics • • • • • • • Assumptions: E + S = ES = E + P The reaction ES=E + P determines catalytic rate The reaction ES=E + P is irreversible ES is in equilibrium. Formation = Removal [E]T = [E] + [ES] When [E]T = [ES], reaction rate is maximum (Vmax) and Vmax = k2[E]T • [E] <<< [S]; so [S] >>> [ES] (can ignore amount of S as ES) 2 Significance of MM equation • KM has a unit of concentration. It is a constant for every enzyme under specific conditions • When [S] <<< KM, Vo is directly proportional to [S] • When [S] >>> KM, Vo = Vmax • When [S] = KM, Vo = ½ Vmax • The KM value of an enzyme indicates the concentration of the substrate required for significant catalysis • Since KM = k2+k-1/k1, when k-1 >>> k2, KM = k-1/k1 • Dissociation constant of ES, KES = [E][S] / [ES] = k-1/k1 • When k-1 >>> k2, KM = KES. High KM = high dissociation = weak binding between E and S • When [E]T = [ES], Vmax = k2[E]T. k2 is called the turnover number: the rate when enzyme is saturated with substrate Allosteric Enzymes • Allosteric enzymes have one or more allosteric sites • Allosteric sites are binding sites distinct from an enzyme’s active site or substrate-binding site • Molecules that bind to allosteric sites are called effectors or modulators • Binding to allosteric sites alters the activity of the enzyme. This is called cooperative binding. Allosteric enzymes display sigmoidal plot of Vo vs [S] • Effectors may be positive or negative • Effectors may be homotropic or heterotropic • Regulatory enzymes of metabolic pathways are allosteric enzymes (eg: feedback inhibition) Line-Weaver Burk Plot • Also called the double-reciprocal plot • 1/Vo = (KM/Vmax).(1/[S]) + 1/Vmax • This is equation for a straight line y = mx + c • Slope = KM/Vmax • Y-intercept = 1/Vmax • X-intercept = -1/KM • Useful for experimental determination of KM and Vmax Enzyme Inhibitors • Specific enzyme inhibitors regulate enzyme activity and help us understand mechanism of enzyme action. (Denaturing agents are not inhibitors) • Irreversible inhibitors form covalent or very tight permanent bonds with aa at the active site of the enzyme and render it inactive. 3 classes: groupspecific reagents, substrate analogs, suicide inhibitors • Reversible inhibitors form an EI complex that can be dissociated back to enzyme and free inhibitor. 3 groups based on their mechanism of action: competitive, non-competitive and uncompetitive. 3 Competitive inhibitors • Compete with substrate for binding to enzyme • E + S = ES or E + I = EI . Both S and I cannot bind enzyme at the same time Non-competitive Inhibitors • Inhibitor binding site is distinct from substrate binding site. Can bind to free enzyme E and to ES • E + I = EI, ES + I = ESI or EI + S = ESI • In presence of I, the equilibrium of E + S = ES is shifted to the left causing dissociation of ES. • Both EI and ESI are enzymatically inactive • This can be reversed / corrected by increasing [S] • Reaction of unaffected ES proceeds normally • Vmax is not changed, KM is increased by (1 + I/Ki) • Inhibition cannot be reversed by increasing [S] • Eg: AZT, antibacterial sulfonamides, the anticancer agent methotrexate etc • KM is not changed, Vmax is decreased by (1 + I/Ki) Uncompetitive Inhibitors • The inhibitor cannot bind to the enzyme directly, but can only bind to the enzyme-substrate complex. • ES + I = ESI. • Both Vmax and KM are decreased by (1+I/Ki). • The effective functional [E] (and [S]) is reduced Irreversible Inhibitors • Group-specific: covalently modify specific side chains of aa in active site of enzyme. Eg: DIPF reacts with serine, iodoacetamide modifies cys • Some substrates transiently covalently modify the enzyme. Substrate analogs are structurally similar to such substrates. Since they are not actually substrates, the enzymatic reaction cannot proceed. • Suicide inhibitors are modified substrates that trick the enzyme into initiating the reaction. Instead of product, reactive intermediates are released which react with the enzyme causing its inactivation 4 Transition State Analogs • Enzyme action may involve conversion of substrate to a temporary transition state. • The transition state of substrate has greater affinity for enzyme than native substrate stabilizing the ES complex and catalysis. • Thus, analogs of substrate transition-state are potent and specific enzyme inhibitors • Due to selective binding of enzyme to the substrate transition state, antibodies produced using transition state analogs as antigens have catalytic properties. Vitamins and Coenzymes • Most enzymes need cofactors for catalytic activity • Coenzymes are enzyme cofactors derived from water-soluble B-complex vitamins. • Vitamins are biomolecules required in minute amounts as dietary supplements. • Fat-soluble vitamins are important for blood clotting (K), Ca and P metabolism (D), vision (A) and as an antioxidant (E). 5