(BIOC 231) Enzyme Kinetics Dr. Ayat B. Al-Ghafari

Learning Objectives By the end of the lecture, the student should be able to : Define Enzyme kinetics Describe the several types of reaction order Know the Michaelis-Menten equation Explain the several modification models for M-M equation

Enzyme Kinetics Study of reaction rate and how they changes in response to change in experimental parameter is known as kinetics Amount of substrate present is one of the key factor affecting the rate of reaction catalyzed by an enzyme in vitro

Low of Mass Action

Zero Order Reaction The rate is independent of the concentration of any of the reactants

First Order Reaction

Second Order Reaction

Michaelis-Menten Kinetics The quantitative theory of enzyme kinetics was proposed by two scientists Leonor Michaelis and Maud Leonora Menten in 1913 Enzyme reactions in Michaelis–Menten kinetics theory occur in two stages: The substrate binds reversibly to the enzyme, forming the enzyme-substrate complex. This is sometimes called the Michaelis-Menten complex The enzyme then catalyzes the chemical step in the reaction and releases the product (Briggs & Haldane)

Kinetics of single-substrate enzyme-catalyzed reactions The relationship between initial velocity and substrate concentration was first suggested by Michaelis-Menten in 1913 The general equation is: k1 k2 E + S ES E + P k-1 Where, S is the substrate E is the enzyme K1, k-1 and k2 are the rate constants

Effect of Substrate Concentration on Reaction Velocity

The Briggs- Haldane modification of M-M equation Briggs & Haldane introduced a more generally valid assumption in 1925 Steady-State assumption: [ES] and [E] are low compared to [S] Then, the rate of change of [ES] would be negligible compared to the rate of change of [P] over initial reaction period Once ES complex is formed, it is maintained at steady state: ES would be broken down as fast as it is being formed k1 k2 E + S ES E + P k-1

Lineweaver-Burk (double reciprocal) plot A linear representation is more accurate and convenient for determining Vmax and Km This equation is obtained by taking reciprocal of both the side of Michelis-Menton equation (or Briggs- Haldane modification) 1/[S] vs. 1/Vo

Lineweaver-Burk (double reciprocal) plot

Lineweaver-Burk (Double Reciprocal) Plot

What does enzyme kinetic mean? Enzyme rates depend on two major factors: Solution conditions Substrate concentration To find the maximum speed of an enzymatic reaction, the substrate concentration is increased until a constant rate of product formation is seen (saturation curve) Saturation happens because, as substrate concentration increases, more and more of the free enzyme is converted into the substrate-bound ES complex

SATURATION CURVE

What does enzyme kinetic mean? At the maximum reaction rate (Vmax) of the enzyme, all the enzyme active sites are bound to substrate, and the amount of ES complex is the same as the total amount of enzyme There are other several important kinetic parameters The Michaelis-Menten constant (Km), which is the substrate concentration required for an enzyme to reach one-half its maximum reaction rate Each enzyme has a characteristic Km for a given substrate

What does enzyme kinetic mean? kcat, also called the turnover number, which is the maximum number of substrate molecules which can be converted to products per molecule of enzyme per unit time (usually in seconds) The efficiency of an enzyme (the specificity constant) can be expressed in terms of kcat/Km

References Harvey, R., and Ferrier, D. (2011) Lippincott’s Illustrated Reviews: Biochemistry 5th edition. Murray, R. et al., (2012) Harper’s Illustrated Biochemistry 29th edition.