Lecture 2 Enzyme Kinetics PDF

Lecture 2 Enzyme Kinetics  Enzymes Enzymes are highly effective catalysts that carry out complex chemical transformations under mild conditions (water, neutral pH). Enzymes show great specificity with regard to the reactions they catalyze and the substrates they r...

Lecture 2 Enzyme Kinetics  Enzymes Enzymes are highly effective catalysts that carry out complex chemical transformations under mild conditions (water, neutral pH). Enzymes show great specificity with regard to the reactions they catalyze and the substrates they react with. Enzymes can be regulated. Enzymes carry out their catalytic role by binding the substrate to a specific area of the protein called the active site. (Companion: Enzymes/Enzyme Kinetics). Several amino acid side chains comprise the active site. Enzymes are Biological Catalysis A catalyst is a substance that increases the rate (velocity) of a chemical reaction. Most biological catalysts are proteins. The material acted upon by the catalyst is the substrate. Although a catalyst participates in the reaction process, it is unchanged after the process is complete. A catalyst increases the rate at which a reaction reaches equilibrium but does not alter Keq or ΔG o' for the reaction. A thermodynamically favourable process s is not made more favourable by the presence of a catalyst. A thermodynamically unfavourable process is not made favourable by the presence of a catalyst.  Catalysis A catalyst functions by lowering the activation energy for a reaction by amount = ΔGo‡. A catalyst does not alter the ΔG for the reaction. ΔGo‡ = ΔHo‡ -TΔSo‡ - a catalyst can accelerate a reaction by affecting either ΔHo‡ or ΔSo‡, or both. Strong binding of the transition state to the catalyst lowers ΔHo‡ - makes it more negative. Proximity and orientation of the substrates on the catalyst favor formation of the transition state by reducing ΔSo‡.  Reaction Rate Theory What determines the rate of a reaction? For every reaction there is a high energy transition state through which the reactants must pass in order for the reaction to occur. The height of the energy barrier, ΔGo‡, determines the rate of the reaction. k=Qe(-ΔGo‡/RT) Q is a collection of constants. # R = the gas constant with a value of 8.314 J K-1mol-1 # T = Temperature of the reaction in Kelvin  Enzyme Kinetics is the study of the rate at which compounds react.  Rate of enzymatic reaction is affected by  enzyme  substrate  effectors  temperature How to Do Kinetic Measurements ?  Michaelis-Menten Equation Derivation Rate of ES formation = k1([ET] - [ES])[S] (where [ET] is total concentration of enzyme E and k-2 is considered negligible. Rate of ES breakdown to product = k-1[ES] + k2[ES]  Plotting Kinetic Data The Michaelis-Menten equation describes a rectangular hyperbola. The enzyme is characterized by two constants: KM and Vmax  Vmax is the maximal rate of the reaction which occurs when [S] >> KM  KM is the substrate concentration that gives 1/2 maximal velocity. What equation models this behaviour? Michaelis-Menten Equation  Meaning of Vmax and Km  An important relationship that can be derived from the Michaelis-Menten equation is the following: If vo is set equal to 1/2 Vmax, then the relation Vmax /2 = Vmax[S]/Km + [S] can be simplified to Km + [S] = 2[S], or Km = [S]. *This means that at one half of the maximal velocity, the substrate concentration at this velocity will be equal to the Km.  For determination of KM and Vmax a linear transformation, the Lineweaver-Burk plot, is useful.  The simplest enzyme mechanism involves the following two steps: The rate of the enzymatic reaction is: v = k1[ES]  Kinetics The Rate Constant For the irreversible reaction A → B. This is a first order reaction (there is only a single reactant). The velocity (v) or reaction rate is given by the rate of formation of product or the rate of disappearance of reactant. The velocity (v) or reaction rate is, where k= the rate constant. For the reaction A + B → products, v = k[A][B]. This is a second order reaction (there are two reactants).  In order to derive a useful equation describing this reaction we make the steady-state assumption, which assumes that over most of the reaction course [ES] is small and does not change, i.e., d[ES]/dt=0. Using this assumption, one can derive the Michaelis-Menten equation. Turnover Number The turnover number of an enzyme, kcat, is the maximal velocity per enzyme molecule per unit of time. Enzyme Efficiency We can re-write the rate equation as: When [S]