The Behavior of Proteins: Enzymes Chapter 6

Enzymes are effective biological catalysts Enzyme: a biological catalyst that can speed up the rate of a chemical reaction Can increase the rate of a reaction by a factor of up to 1020 over uncatalyzed reactions RNAs (ribozymes) Globular proteins some enzymes are so specific that they catalyze the reaction of only one stereoisomer; others catalyze a family of similar reactions

Thermodynamics and Kinetics of Reaction Thermodynamics – whether a reaction is spontaneous or not Kinetics – determines how fast a reaction occurs If a reaction is involving many catalysts/enzymes then it is spontaneous – activation energy is negative. Even if it is negative sometimes, reactions might not happen quickly

What is Activation Energy? The difference between energies of reactants and products – Standard free energy = ΔºG The rate of a reaction depends on its activation energy = ΔºG+ Speed up reactions Do not alter free energy change Lower the activation energy The difference between energies of reactants (initial state) and energies of the products (final state) of a reaction gives energy change for that reaction – standard free energy change ΔºG

What is Activation Energy? Energy required to initiate a reaction ΔºG for an uncatalyzed reaction is higher than that of catalyzed reaction Uncatalyzed reactions requries more energy to get started and is slower in progress than a catalyzed reaction. Enzymes reduce activation energy. The activation energy can be seen as the amount of free energy required to bring the reactants to transition state.

Enzyme Catalysis Consider the reaction Conversion of H2O2 to water and oxygen is an example of effect of catalysis on activation energy. The energy is lowered if the reaction is allowed to proceed on platinum surfaces but is lowered even more by enzyme catalase

Temperature dependence of catalysis Temperature can also catalyze reaction Increasing temperature will eventually lead to protein denaturation Temperature can increase rate of reaction

Kinetic equations of enzymatic reactions For any reaction The rate of reaction is given by rate equation A and B are reactants and P is the product. The rate of reaction can be expressed as rate of disappearance of one of the reactants or appearance of product. Or the negative signs indicates that A and B are being used up to produce P. K is proportional to product of concentrations of reactants. raised to appropriate powers. K, f and g should be determined experimentally Where k is a proportionality constant called the specific rate constant

How Enzymes bind to Substrate? In an enzyme-catalyzed reaction Substrate, S: a reactant Active site: the small portion of the enzyme surface where the substrate(s) becomes bound by noncovalent forces, e.g., hydrogen bonding, electrostatic attractions, Van der Waals attractions The binding of substrate to enzyme occurs because of interactions between substrate and and the side chains and backbone groups of amino acids making up an active site

What are the two Binding Models? Two models have been developed to describe formation of the enzyme-substrate complex Lock-and-key model: Substrate binds to that portion of the enzyme with a complementary shape Induced fit model: Binding of the substrate induces a change in the conformation of the enzyme that results in a complementary fit

2 Models of E-S Complex Formation

Formation of Product – Figure 6.5 Product formed should be released for more substrate to come and bind to the enzyme to form more products

Chymostrypsin - An Example of Enzyme Catalysis Chymotrypsin catalyzes The selective hydrolysis of peptide bonds where the carboxyl is contributed by Phe and Tyr It also catalyzes hydrolysis of the ester bonds

Non-Allosteric Enzyme Behavior Point at which the rate of reaction does not change Enzyme is saturated Maximum rate of reaction is reached Hyperbolic

Allosteric Enzyme Behavior Sigmoidal shape- characteristic of allosterism

Michaelis-Menten Kinetics Initial rate of an enzyme-catalyzed reaction versus substrate concentration

What is Vmax and KM? Vmax – describes velocity of an enzyme-catalyzed reaction when there is a saturating level of substrate Determines the individual rate constant (Kp) KM is equal to substrate concentration that generates half of Vmax.

Lineweaver-Burk Plot KM is the dissociation constant for ES; the greater the value of KM, the less tightly S is bound to E Vmax is the turnover number Reciprocal plot of velocity versus substrate.

Turnover Numbers • Vmax is related to the turnover number of enzyme:also called kcat • Number of moles of substrate that react to form product per mole of enzyme per unit of time

What is an enzyme inhibitor? A substance that interferes with action of an enzyme and slows rate of reaction – Enzyme inhibitor

Reversible Enzyme Inhibition Reversible inhibitor: a substance that binds to an enzyme to inhibit it, but can be released Competitive inhibitor: binds to the active (catalytic) site and blocks access to it by substrate

Reversible Enzyme Inhibition Noncompetitive inhibitor: binds to a site other than the active site; inhibits the enzyme by changing its conformation

Non-reversible enzyme inhibition Irreversible inhibitor: a substance that causes inhibition that cannot be reversed usually involves formation or breaking of covalent bonds to or on the enzyme

Other types of Inhibition Uncompetitive- inhibitor can bind to the ES complex but not to free E. Vmax decreases and KM decreases. Mixed- Similar to noncompetitively, but binding of I affects binding of S and vice versa

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