Multiple Alternative Enzyme-Substrate Reactions

As a consequence, the developed theory of allosteric regulation (fi-om Greek alios, other + stereos, space) prescribes that, within a cooperative interaction, the binding of one ligand (substrate) at a specific site is influenced by the binding of another ligand (inhibitor) at a different or allosteric site on the protein (or enzyme). However, actually, such behavior 

Basically, an alternative -substrate system consists of -reactions of the Michaelis-Menten type (1.4) 

The associated conservation laws now look as generalizations of the basic ones given in Eqs. (1.9) and (1.10)  

Following the deduction line of the foreground enz5mies kinetic, the specific Michaelis-Menten /-constants 

Additionally, a few new notations are considered here (Schnell Mendoza, 2000b), namely the first order rate i-comtants. 

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Drugs containing multiple functional groups are substrates for multiple drug-metabolizing enzymes and pathways. The N-demethylation vs O-dealkylation of ethylmorphine (Scheme 11.13) demonstrates that one reaction pathway may predominate. Propranolol is an example of a compound that forms multiple alternative metabolites (Scheme 11.14). 

For the purpose of the present discussion the term transient kinetics is applied to the time course of a reaction from the moment when enzyme and substrate are mixed, t=0, until either a steady state or equilibrium is established. The difference between the kinetic problems discussed in section 3.3 and in the present section is, respectively, the presence of catalytic as distinct from catalytic concentrations of enzyme. Here we are concerned with the stoichiometry of enzyme states. Transient kinetic experiments with enzymes can be divided into two types. The first of these (multiple turnover) is carried out under the condition that the initial concentrations of substrate and enzyme are Cs(0) Ce(0) and c it) can, therefore, be regarded as constant throughout the course of the reaction until a steady state is attained. Alternatively, in a single turnover reaction, when Cs(0)reaction intermediates is observed until the overall process is essentially complete. These two possibilities will be illustrated with specific examples. In connection with a discussion of the approach to the steady state, in section 3.3 it was emphasized that, at t = 0, the concentrations of the intermediates, enzyme-substrate and enzyme-product complexes, are zero and, therefore, the rate of product formation is also zero. Under the experimental conditions used for steady state rate measurements and for enzyme assays, the first few seconds after the initiation of a reaction are ignored. However, when the experimental techniques and interpretation discussed below are used, events during the first few milliseconds of a reaction can be analysed and provide important information. With suitable monitors it is possible to follow the formation and decay of enzyme complexes with substrates and... 

A titrametric assay of PLCSc, alternatively called the pH-stat method, was the workhorse in early studies [28]. This method simply involves titrating the acidic product of the PLC reaction as it is formed with a solution of standard base. An advantage of this continuous assay is that it can be used to detect the turnover of both synthetic and natural substrates, and its sensitivity has been estimated to be in the 20-100 nmol range. However, the pH-stat assay has low throughput capability, and it cannot be easily performed in a parallel fashion with multiple substrate concentrations. It is also necessary to exclude atmospheric carbon dioxide from the aqueous media containing the enzyme and substrate. 

The process of identifying the products of the interaction between the enzyme and alternate substrate depends a great deal on the inhibitor itself. If the compound contains a chromophore or fluorophore, changes in the absorbance or fluorescence spectra with the addition of enzyme can be monitored and used to identify products (Krantz et al., 1990). For multiple product reactions, single turnover experiments can be used to determine relative product distribution. Stoichiometric quantities of enzyme and inhibitor can be incubated for full inhibition, followed by the addition of a rapid irreversible inhibitor of the enzyme, such as an affinity label. This will act as a trap for enzyme as the enzyme-inhibitor complex breaks down. Analysis of the products will determine relative... 

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