Structure enzyme: coenzyme: inhibitor complex

Coenzyme and substrate analogs. The structures of enzyme NAD1 substrate complexes (Fig. 15-3) may be studied by X-ray crystallography under certain conditions or can be inferred from those of various stable enzyme-inhibitor complexes or from enzyme reconstituted with NAD+ that has been covalently... 

A crystal structure of a ternary complex of horse liver alcohol dehydrogenase with NADH and the inhibitor, dimethyl sulfoxide, first at 4.5 A resolution1365 and a further refinement to 2.9 A resolution,1366 has been published by Eklund et al. The gross structure of the ternary complex is similar to that of the free enzyme structure. Each subunit is divided into a coenzyme-binding domain and a catalytic domain. The subunits are joined together near the... 

Kinetic studies of reversible inhibition by substrate analogs give evidence of the mode of action of the inhibitor and the types of enzyme-inhibitor complex formed, and estimates of their dissociation constants. The complexes may be isolated and sometimes crystallized. Studies of the stabilities, optical properties, and structures of ternary complexes of enzymes, coenzymes, and substrate analog in particular, as stable models of the catalytically active ternary complexes or of the transition state for hydride transfer (61,79,109,115-117), can only be touched upon here there is direct evidence with several enzymes that the binding of coenzymes is firmer in such complexes than in their binary complexes (85,93,118), which supports the indirect, kinetic evidence already mentioned for a similar stabilization in active ternary complexes. 

Substrate binding also induces a conformational change in this enzyme. When both coenzymes and substrate bind the "closed" conformation of the enzyme is formed by a rotation of the catalytic domains of the two subunits relative to the coenzyme-binding do-mains.50 51a Structures of ternary complexes with inhibitors and with substrates have also been established. 

Another enzyme for which X-ray diffraction studies have aided in an analysis of the mode of action is the enzyme dihydrofolate reductase. This catalyzes the reduction of 7,8-dihydrofolate to 5,6,7,8-tetrahydrofolate, an essential coenzyme used in the synthesis of thymidylate, inosinate, and methionine. The antitumor agent methotrexate is a powerful inhibitor of dihydrofolate reductase, causing, on binding, a cellular deficiency of thymidylate (the cause of its antitumor activity). The crystal structures of the enzyme from two bacterial sources—Escherichia coli and Lactobacillus casei—and from chicken liver have been studied (88-90). Both the E. coli and L casei enzymes have been studied as complexes with methotrexate bound at the active site, and, in the case of the . casei enzyme, the cofactor, NADPH, was also present. 

However, the findings (171) that binary complexes of zinc-free enzyme and coenzyme bind substrates and substrate competitive inhibitors such as isobutyramide cannot be taken as evidence that zinc does not participate in the catalytic action. Several SH groups are probably oxidized in the zinc-free enzyme (170). Evidence has also been presented (172) of other structural differences compared to the catalytically active enzyme. Thus, artificial binding to this enzyme with no relevance to the catalytic action is not unlikely. 

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