Rationale for Transient Kinetic Analysis

The kinetic analysis of an enzyme mechanism often begins by analysis in the steady state therefore, we first consider the conclusions that can be derived by steady-state analysis and examine how this information is used to design experiments to explore the enzyme reaction kinetics in the transient phase. It has often been stated that steady-state kinetic analysis cannot prove a reaction pathway, it can only eliminate alternate models from consideration (5). This is true because the data obtained in the steady state provide only indirect information to define the pathway. Because the steady-state parameters, kcat and K, are complex functions of all of the reactions occurring at the enzyme surface, individual reaction steps are buried within these terms and cannot be resolved. These limitations are overcome by examination of the reaction pathway by transient-state kinetic methods, wherein the enzyme is examined as a stoichiometric reactant, allowing individual steps in a pathway to be established by direct measurement. This is not to say that steady-state kinetic analysis is without merit rather, steady-state and transient-state kinetic studies complement one another and analysis in the steady state should be a prelude to the proper design and interpretation of experiments using transient-state kinetic methods. Two excellent chapters on steady-state methods have appeared in this series (6, 7) and they are highly recommended. 

With this perspective, one can summarize the three things learned by steady-state kinetic analysis as follows  

The order of binding of substrates and release of products serves to define the reactants present at the active site during catalysis it does not establish the kinetically preferred order of substrate addition and product release or allow conclusions pertaining to the events occurring between substrate binding and product release. 

The value of jtcat sets a lower limit on each of the first-order rate constants governing the conversion of substrate to product following the initial collision of substrate with enzyme. These include conformational changes in the enzyme-substrate complex, chemical reactions (including the formation and breakdown of intermediates), and conformational changes that limit the rate of product release. 

The value of k aJKm defines the apparent second-order rate constant for substrate binding and sets a lower limit on the true second-order rate constant for substrate binding. The term k at/Km is less than the true rate constant by a 

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