Enzymes multiple substrates

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

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. 

All enzymatic reactions are initiated by formation of a binary encounter complex between the enzyme and its substrate molecule (or one of its substrate molecules in the case of multiple substrate reactions see Section 2.6 below). Formation of this encounter complex is almost always driven by noncovalent interactions between the enzyme active site and the substrate. Hence the reaction represents a reversible equilibrium that can be described by a pseudo-first-order association rate constant (kon) and a first-order dissociation rate constant (kM) (see Appendix 1 for a refresher on biochemical reaction kinetics) ... 

The three bi-bi mechanisms described here provide some sense of the diversity of mechanisms available to enzymes that act on multiple substrates. This is by no... 

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. 

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. 

Substrates may affect enzyme kinetics either by activation or by inhibition. Substrate activation may be observed if the enzyme has two (or more) binding sites, and substrate binding at one site enhances the alfinity of the substrate for the other site(s). The result is a highly active ternary complex, consisting of the enzyme and two substrate molecules, which subsequently dissociates to generate the product. Substrate inhibition may occur in a similar way, except that the ternary complex is nonreactive. We consider first, by means of an example, inhibition by a single substrate, and second, inhibition by multiple substrates. 

Similar to irreversible reactions, biochemical interconversions with only one substrate and product are mathematically simple to evaluate however, the majority of enzymes correspond to bi- or multisubstrate reactions. In this case, the overall rate equations can be derived using similar techniques as described above. However, there is a large variety of ways to bind and dissociate multiple substrates and products from an enzyme, resulting in a combinatorial number of possible rate equations, additionally complicated by a rather diverse notation employed within the literature. We also note that the derivation of explicit overall rate equation for multisubstrate reactions by means of the steady-state approximation is a tedious procedure, involving lengthy (and sometimes unintelligible) expressions in terms of elementary rate constants. See Ref. [139] for a more detailed discussion. Nonetheless, as the functional form of typical rate equations will be of importance for the parameterization of metabolic networks in Section VIII, we briefly touch upon the most common mechanisms. 

Figure 9.2. The inherent metabolic flexibility of the isoprenoid pathway leading to the synthesis of some carotenoid pigments. Genes coding for two enzymes capable of acting on carotenoid structures were introduced into Escherichia coli which had already been transformed to give it the capacity to make p,p-carotene. Both of the two introduced new enzymes (one shown with red arrows and the other with blue arrows) acted on multiple substrates because of their lack of specificity. The resulting matrix of transformations means that nine different products can be made by just two tailoring enzymes. (Adapted from Umeno et al. ° who used data from Misawa et al. °)...

The investigations carried out by Professor French and his students were based on sound experimental approaches and on intuitive theoretical considerations. The latter often resulted in new experiments for testing a hypothesis. On the basis of theoretical considerations, Professor French proposed a model for the structure of the amylopectin molecule, and the distribution of the linear chains in this molecule. This model was tested by utilizing enzymes that selectively cleave the linear chains, and the results substantiated the theoretical deductions. He proposed a theory on the nature and types of reactions occurring in the formation of the enzyme - starch complex during the hydrolysis of starch by amylases. In this theory, the idea of multiple attack per single encounter of enzyme with substrate was advanced. The theory has been supported by results from several types of experiments on the hydrolysis of starch with human salivary and porcine pancreatic amylases. The rates of formation of products, and the nature of the products of the action of amylase on starch, were determined at reaction conditions of unfavorable pH, elevated temperatures, and increased viscosity. The nature of the products was found to be dramatically affected by the conditions utilized for the enzymic hydrolysis, and could be accounted for by the theory of the multiple attack per single encounter of substrate and enzyme. 

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. 

The decisive experiment of Biebricher and Luce is shown in Fig. 4. A synthesis medium containing highly purified enzyme and substrates is incubated and maintained at a suitable temperature, for a time adequate to allow the multiplication of any templates present but too short to enable products to arise de novo. Then the solution is divided into portions. Each portion is incubated long enough to allow synthesis de novo and the products are compared by the fingerprint method. If the impurity hypothesis is correct, then multiplication of the impurity in the first phase should lead to the same product from each portion of the incubated medium. If the de novo hypothesis is correct then the products should be different, since at the beginning different enzyme molecules were working on different products. Selection, that is, preferential reproduction of one rudimentary strand, could not yet take place, since in the first, short incubation none of the products de novo was complete. 

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. 

Enzymes often require multiple substrates to complete their catalytic cycle. This may involve combining two compounds into one molecule or transferring atoms or electrons from one substrate to another. The substrates may both bind to an enzyme and react collectively, or each substrate might bind, react, and release sequentially. With two substrates, if both bind to the enzyme, a ternary complex (ES S2) will form (Scheme 4.8). The order of substrate addition may be important (ordered) or not (random order). Cases in which the two substrates react sequentially follow a double-displacement, or ping-pong, mechanism (Scheme 4.9). Enzymes requiring more than two substrates have more complicated complexation pathways. 

Multiple substrate mechanisms follow Michaelis-Menten kinetics. Experiments are performed with constant concentrations of the enzyme and one substrate with variation of the second substrate concentration ([S2]). (Note that the second substrate concentration [S2] is not the same as a deceptively similar term, the square of the substrate concentration [S]2.) Plotting V against [S2] gives a hyperbolic curve and allows determination of Km for the second substrate. The Km values for all substrates may be found in a similar fashion. 

Enzymes that catalyze redox reactions often require a coenzyme such as NAD+ or FADH2 in addition to a substrate. These are all multiple substrate enzymes. Each substrate and coenzyme will have its own Km value. The substrates for glutamate dehydrogenase, an enzyme with three substrates in both forward and reverse directions, are shown in Scheme 4.10 with their K values.9... 

As most NRPS multienzymes are multidomain proteins with multiple activation domains, multiple sites may participate in the reactions assayed, and no clear result concerning a single specific site may result. In ACV synthetases, the nonadditivity of the initial rates has been observed in the S. clavuligerus enzyme [35] and the A. chrysogenum enzyme [1]. Two or more site activations of one substrate amino acid could be expected to depend on different binding constants, and thus be detectable by kinetic analysis. So far, however, no evidence for mixed types of concentration dependence has been found. It is thus not yet clear if nonadditivity results from misactivation or alteration of kinetic properties in the presence of multiple substrates. In the case of gramicidin S synthetase 2, evidence for misactivations has been reported [59],... 

If enzyme activation and the other unusual kinetic characteristics result from multiple substrates in the active site, kinetic parameters will be difficult to characterize and drug interactions will be more difficult to predict, since they are a function of the enzyme and of both the substrates. In addition, there are some indications that non-Michaelis-Menten kinetics can be seen in vivo (27-29). 

If non-Michaelis-Menten kinetics for all P450 enzymes are a result of multiple substrates binding to the enzyme, then the reaction kinetics for the binding of two substrates to an active site can be complicated. A number of analyses of... 

In retrospect, Cushny was one of the earliest investigators to point out the diastereoisomeric situations which occur when a chiral biopolymer interacts with the enantiomeric forms of another molecule. The three-point attachment and polyaffinity concepts provided an easily visualized picture of how the differentiation might occur. For a time, unfortunately, the possibility of differentiation by a one-point approach was not clearly recognized and three-point attachment became somewhat dogmatic. With more detailed structural investigations, it is becoming clear that interactions between enzymes and substrates, and receptors and drugs, often involve a multiplicity of interactions. 

Previous sections of this chapter have focused on developing general principles for enzyme-catalyzed reactions based on analysis of single-substrate enzyme systems. Yet the majority of biochemical reactions involve multiple substrates and products. With multiple binding steps, competitive and uncompetitive binding interactions, and allosteric and covalent activations and inhibitions possible, the complete set of possible kinetic mechanisms is vast. For extensive treatments on a great number of mechanisms, we point readers to Segel s book [183], Here we review a handful of two-substrate reaction mechanisms, with detailed analysis of the compulsory-order ternary mechanism and a cursory overview of several other mechanisms. 

CYP enzymes are a large and diverse superfamily of haemoproteins. Primarily membrane-associated proteins, they are located in the inner membrane of mitochondria and the endoplasmic reticulum of cells, and metabolise thousands of endogenous and exogenous compounds. Most of these enzymes can metabolise multiple substrates, and many can catalyse multiple reactions. While prevalent in the liver, CYP enzymes are also present in most other tissues of the body, and play an important role in hormone synthesis and breakdown (including oestrogen and testosterone synthesis and metabolism), cholesterol synthesis and vitamin D metabolism. Hepatic CYPs are the most widely smdied. 

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... 

The other extreme is when a compound binds only to the E S complex but not to the free enzyme, in which case uncompetitive inhibition occurs (Scheme 2). Although it is rare in single substrate reactions, it is common in multiple substrate systems. An inhibitor of a two-substrate enzyme that is competitive against one of the substrates often is found to give uncompetitive inhibition when the other substrate is varied. The inhibitor binds at the active site but only prevents the binding of one of the substrates. 

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