Enzyme reactions multiple, enzymes/substrates

In a complex enzyme reaction, multiple substrate-enzyme complexes are formed. Assume the following reaction mechanisms are taking place in three consecutive stages ... 

During the enzyme reaction of a substrate, multiple metabolite isomers may be formed. Thus, a baseline LC separation of the metabolites is desired. For example, more than five hydroxylated testosterone isomers (2-, 6-, 15-, and 16-positions) were seen after an incubation of testosterone with HLM. BDS Hypersil C8 was successfuly used for a separation of the five isomers (Table 16.1). Majority of the metabolites (>82%) was due to the 6 -OH-testosterone. Similarly, two hydroxylated isomers of midazolam at 1 and 4 -positions were also isolated with the given HPLC condition. 

Enzyme Immunosensors. Enzyme immunosensors are enzyme immunoassays coupled with electrochemical sensors. These sensors (qv) require multiple steps for analyte determination, and either sandwich assays or competitive binding assays maybe used. Both of these assays use antibodies for the analyte of interest attached to a membrane on the surface of an electrochemical sensor. In the sandwich assay type, the membrane-bound antibody binds the sample antigen, which in turn binds another antibody that is enzyme-labeled. This immunosensor is then placed in a solution containing the substrate for the labeling enzyme and the rate of product formation is measured electrochemically. The rate of the reaction is proportional to the amount of bound enzyme and thus to the amount of the analyte antigen. The sandwich assay can be used only with antigens capable of binding two different antibodies simultaneously (53). 

In general, enzymes are proteins and cany charges the perfect assumption for enzyme reactions would be multiple active sites for binding substrates with a strong affinity to hold on to substrate. In an enzyme mechanism, the second substrate molecule can bind to the enzyme as well, which is based on the free sites available in the dimensional structure of the enzyme. Sometimes large amounts of substrate cause the enzyme-catalysed reaction to diminish such a phenomenon is known as inhibition. It is good to concentrate on reaction mechanisms and define how the enzyme reaction may proceed in the presence of two different substrates. The reaction mechanisms with rate constants are defined as ... 

In what follows, enzyme reactions are treated as if they had only a single substrate and a single product. While most enzymes have more than one substrate, the principles discussed below apply with equal vaUdity to enzymes with multiple substrates. 

Although the Michaelis-Menten equation is applicable to a wide variety of enzyme catalyzed reactions, it is not appropriate for reversible reactions and multiple-substrate reactions. However, the generalized steady-state analysis remains applicable. Consider the case of reversible decomposition of the enzyme-substrate complex into a product molecule and enzyme with mechanistic equations. 

Most, if not all, milks contain sufficient amounts of lipase to cause rancidity. However, in practice, lipolysis does not occur in milk because the substrate (triglycerides) and enzymes are well partitioned and a multiplicity of factors affect enzyme activity. Unlike most enzymatic reactions, lipolysis takes place at an oil-water interface. This rather unique situation gives rise to variables not ordinarily encountered in enzyme reactions. Factors such as the amount of surface area available, the permeability of the emulsion, the type of glyceride employed, the physical state of the substrate (complete solid, complete liquid, or liquid-solid), and the degree of agitation of the reaction medium must be taken into account for the results to be meaningful. Other variables common to all enzymatic reactions—such as pH, temperature, the presence of inhibitors and activators, the concentration of the enzyme and substrate, light, and the duration of the incubation period—will affect the activity and the subsequent interpretation of the results. 

Still another possibility is that the inhibitor binds only to the enzyme-substrate complex and not to the free enzyme (fig. 7.14c). This reaction is called uncompetitive inhibition. Uncompetitive inhibition is rare in reactions that involve a single substrate but more common in reactions with multiple substrates. Plots of 1/v versus 1/[S] at different concentrations of an uncompetitive inhibitor give a series of parallel lines. 

We now return to the dimensionless pH-dependent reaction term 9Ipn in (2.28). Enzymes are proteins that are subject to multiple protonation equilibria. In that respect, they are polyelectrolytes. The scheme shown in Fig. 2.13 depicts the simplest situation, with only one product-forming pathway in which the product P is formed from the protonated enzyme/substrate complex H+ES. 

The simplest enzymatic system is the conversion of a single substrate to a single product. Even this straightforward case involves a minimum of three steps binding of the substrate by the enzyme, conversion of the substrate to the product, and release of the product by the enzyme (Scheme 4.6). Each step has its own forward and reverse rate constant. Based on the induced fit hypothesis, the binding step alone can involve multiple distinct steps. The substrate-to-product reaction is also typically a multistep reaction. Kinetically, the most important step is the rate-determining step, which limits the rate of conversion. 

Cofactor Requirements and Multiple Enzyme Catalytic Sequences Many enzyme-catalyzed reactions of potential interest for synthetic chemistry require cofactor substrates which serve as electron donors or acceptors or phosphate donors. These chemicals are much too costly to be used as process feedstocks, so economical practice of these cofactor-requiring enzyme-catalyzed reactions necessitates recovery and chemical regeneration of the required cofactor substrate. In spite of significant research aimed at solving this problem, no general solution exists at present. 

Enzyme reactions proceed, in general, via several intermediate states. A simple model incorporating multiple states is shown below enzyme and substrate assodate to form an enzyme-substrate complex, which undergoes a conformational change to ES before breaking down into enzyme and product... 

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