Enzyme kinetics multi substrate reactions

At the start of optimization of the reaction system, suitable values for pH and temperature have to be chosen as a function of the properties of the reactants and enzymes. Fortunately, most enzyme reactions operate in a narrow band with respect to pH value (7-10) and temperature (30-50 °C). The initial substrate concentration and, in the case of two-substrate reactions, the stoichiometric ratio of the two reactants, have to be selected. The selected enzyme concentration influences both the achievable space-time-yield as well as the selectivity in the case of undesired parallel or consecutive side reactions. In the case of multi-enzyme systems, the optimal activity ratio has to be found. The activity and stability of all the enzymes involved have to be known as a function of the reaction conditions, before the kinetic measurements are made. Enzyme stability is an important aspect of biocatalytic processes and should be expressed preferably as an enzyme unit consumption number, with the dimension unit of activity per mass of product (such as mole, lb, or kg). In multi-enzyme systems the stability of all the enzymes has to be optimized so that an optimal reaction rate and space-time-yield result. 

In addition to the reaction sequence shown, the substrate may have to mutarotate non-enzymically to the form favored for affording the first enzyme-substrate complex. In such multi-stage reactions, the observed isotope-effects for the overall reaction are usually low. The situation is further complicated by possible differences in Km between a deuterated substrate and the unlabeled substrate, so that, unless a full kinetic analysis of the reaction is performed, the values determined for the apparent isotope-effect may vary. 

While the majority of these concepts are introduced and illustrated based on single-substrate single-product Michaelis-Menten-like reaction mechanisms, the final section details examples of mechanisms for multi-substrate multi-product reactions. Such mechanisms are the backbone for the simulation and analysis of biochemical systems, from small-scale systems of Chapter 5 to the large-scale simulations considered in Chapter 6. Hence we are about to embark on an entire chapter devoted to the theory of enzyme kinetics. Yet before delving into the subject, it is worthwhile to point out that the entire theory of enzymes is based on the simplification that proteins acting as enzymes may be effectively represented as existing in a finite number of discrete states (substrate-bound states and/or distinct conformational states). These states are assumed to inter-convert based on the law of mass action. The set of states for an enzyme and associated biochemical reaction is known as an enzyme mechanism. In this chapter we will explore how the kinetics of a given enzyme mechanism depend on the concentrations of reactants and enzyme states and the values of the mass action rate constants associated with the mechanism. 

The previous sections may give the impression that it is an easy matter to establish the mechanism of a multi-substrate enzyme. In fact, more often than not uncertainty and controversy surround such mechanisms for many years despite an abundance of experimental work. We have assumed an ideal situation whereas there are a number of possible obstacles in practice. For example the reaction may be effectively irreversible so that it is only possible to measure the kinetic parameters for one direction of reaction the substrate specificity may be so stringent that it is impossible to apply tests which rely on using a range of alternative substrates the available methods of rate measurement may not be sufficiently sensitive to allow all the kinetic parameters to be determined rehably. The last problem at least is one that allows some hope the kinetic study of NAD-dependent dehydrogenases became much more incisive once fluorescence measurement took over from absorbance measurement as the method of choice [52,57]. Nevertheless there is clearly a need for as many criteria of mechanism as may be mustered and the study of inhibition patterns is a valuable adjunct to the methods already discussed. 

This textbook for advanced courses in enzyme chemistry and enzyme kinetics covers the field of steady-state enz5mie kinetics from the basic principles inherent in the Michaelis-Menten equation to the expressions that describe the multi-substrate enzyme reactions. The purpose of this book is to provide a simple but comprehensive framework for the study of enzymes with the aid of kinetic studies of enzyme-catalyzed reactions. The aim of enzyme kinetics is twofold to study the kinetic mechanism of enz5mie reactions, and to study the chemical mechanism of action of enzymes. 

The detailed characterization of the enzyme kinetics of DrrA clearly estab-hshed that Rabl GTP and ATP are the preferred enzyme substrates. The enzymatic parameters were used to conveniently estimate the reaction time for multi-miUigram amounts of homogeneously adenylylated Rabl GTP or the nonhydrolyzable derivative of GTP, Rabl GppNHp (guanosrne-5 -0-[(p,y)-imido] -triphosphate). 

Multi-substrate enzymes (see) catalyse reactions of two or more substrates. Such enzymes can form a number of different complexes (known as enzyme species) with one or both substrates and/or products. The order in which these species are formed may be random or ordered. Cleland s short notation (see) is a convenient way of representing the possibilities. The kinetics of such reactions become extremely complicated enzyme networks (see Enzyme graphs) provide a means of sununarizing them. To evaluate the kinetic data for such systems, one must resort to a computer. Furthermore, the information gained from steady-state experiments may not be sufficient. A number of methods of very rapid measiu ement have been used to investigate the pre-steady-state condition of reactions, including stopped flow, temperature jump and flash methods. 

A further requirement for the development of a multi-enzyme oxidizing process would be the determination of the kinetic parameters of the enzymes and hence development of a model of the intended reaction system in terms of the relative productivities of the enzymes with respect to substrate conversion rates as well as electron transfer stoichiometry. 

Unfortunately, most enzymes do not obey simple Michaelis-Menten kinetics. Substrate and product inhibition, presence of more than one substrate and product, or coupled enzyme reactions in multi-enzyme systems require much more complicated rate equations. Gaseous or solid substrates or enzymes bound in immobilized cells need additional transport barriers to be taken into consideration. Instead of porous spherical particles, other geometries of catalyst particles can be apphed in stirred tanks, plug-flow reactors and others which need some modified treatment of diffusional restrictions and reaction technology. 

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