Enzyme mechanism compulsory-order ternary

A second ternary complex reaction mechanism is one in which there is a compulsory order to the substrate binding sequence. Reactions that conform to this mechanism are referred to as bi-bi compulsory ordered ternary complex reactions (Figure 2.13). In this type of mechanism, productive catalysis only occurs when the second substrate binds subsequent to the first substrate. In many cases, the second substrate has very low affinity for the free enzyme, and significantly greater affinity for the binary complex between the enzyme and the first substrate. Thus, for all practical purposes, the second substrate cannot bind to the enzyme unless the first substrate is already bound. In other cases, the second substrate can bind to the free enzyme, but this binding event leads to a nonproductive binary complex that does not participate in catalysis. The formation of such a nonproductive binary complex would deplete the population of free enzyme available to participate in catalysis, and would thus be inhibitory (one example of a phenomenon known as substrate inhibition see Copeland, 2000, for further details). When substrate-inhibition is not significant, the overall steady state velocity equation for a mechanism of this type, in which AX binds prior to B, is given by Equation (2.16) ... 

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. 

Compulsory-order ternary mechanism Consider first the case where two substrates (A and B) bind to an enzyme in an ordered manner and two products (P and Q) dissociate in an ordered manner as well ... 

This mechanism is known as the ordered bi-bi mechanism ( bi-bi denotes a bi-substrate bi-product reaction), or the compulsory-order ternary mechanism , where the term ternary refers to the three-species complex formed by the binding of two substrates to the enzyme. 

Figure 4.9 Basic compulsory-order ternary-complex mechanism. The basic ordered mechanism for the general reaction A + B P + Q, with a = [A], b = [B], p = [P], and q = [Q] is illustrated. The four states are unbound enzyme (E), enzyme-substrate A complex (E-A), enzyme-substrate A-substrate B complex (E-AB), and enzyme-product Q complex (E-Q).

As an example to illustrate analysis of kinetic data to characterize the mechanism of a real enzyme, here we apply the general compulsory-order ternary mechanism introduced above to citrate synthase to determine kinetic parameters for several isoforms of this enzyme and to elucidate the mechanisms behind inhibition by products and other species not part of the overall chemical reaction. 

It should be re-emphasised that for a given enzyme the compulsory-order mechanism (with or without kinetically significant ternary complexes) is not just a single mechanism, because for a real enzyme the symbols denote real substrates so that there is the question of which is the leading substrate in each direction. Some of the... 

ATP -I- 2 -deoxynucleoside = ADP + 2 -deoxynucleoside 5 -phosphate (<1>, compulsory ordered steady-state reaction mechanism with formation of a ternary complex with the phosphate donor and acceptor [2] the enzyme from embryonic cells of Drosophila melanogaster differs from other deoxynucleoside kinases [EC 2.7.1.76 (deoxyadenosine kinase) and EC 2.7.1.113 (deoxyguanosine kinase)] in its broad specificity for all four common deoxynucleosides)... 

The donor and acceptor can be bound in compulsory order or random order, the main point being that both must be bound at adjacent sites before group transfer can occur. This mechanism is kinetically indistinguishable from one in which an additional covalent phosphoryl-enzyme or nucleotidyl-enzyme exists and connects the two ternary complexes, as in Eq. (3). In this mechanism the enzyme mediates group transfer by nucleophilic catalysis, utilizing an enzymic nucleophile as the catalytic functional group. 

In a sequential mechanism, on the other hand, such as is followed by most NAD(P) -linked dehydrogenases [42], the enzyme forms a ternary complex i.e. a complex containing the enzyme itself and both substrates. This allows for several further possibilities. There may be either random-order or compulsory-order binding. If there is a compulsory order of substrate addition and product release, there are 4 possible sequences ... 

The classical steady-state studies of Theorell and Chance showed that the increased affinity for substrate by the NADH-bound enzyme leads to a distinct sequence of the binding of coenzyme and substrate and subsequent reaction.1442. The binding of coenzyme is a compulsory step prior to substrate binding. Release of products from the enzyme site occurs via reversal of the sequence. This mechanism, known as an ordered bi-bi mechanism because of the required order of association and dissociation of the coenzyme and substrate with ternary complex formation is summarized in Scheme 6, where E, S and P represent enzyme, substrate and product respectively. 

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