Enzyme active conformation

The catalytically active enzyme substrate complex is an interactive structure in which the enzyme causes the substrate to adopt a form that mimics the transition-state intermediate of the reaction. Thus, a poor substrate would be one that was less effective in directing the formation of an optimally active enzyme transition-state intermediate conformation. This active conformation of the enzyme molecule is thought to be relatively unstable in the absence of substrate, and free enzyme thus reverts to a conformationally different state. 

Probing the Active Sites of Enzymes with Conformationally Restricted Substrate Analogs. BY G. L. KENYON AND J. A. FEE, Department of Chemistry, University of California, Berkeley, California. 381... 

Probing the Active Sites of Enzymes With Conformationally Restricted Sutetrate Analogs... 

In order to give useful information about an enzyme, a conformationally restricted active-site-directed analog inhibitor need not bind to the enzyme irreversibly. In a study of the enzyme fructose 1,6-diphosphatase from rabbit liver, Benkovic et al, have investigated the question of the reactive form of the fructose 1,6-diphosphate in the enzymatic process (104,105). Three likely forms are shown in structures 50, 51 and 52. 

So far, it has been established from in vitro studies that the enzyme undergoes phosphorylation, a process that changes the conformation of the enzyme protein and leads to an increase in its activity. This involves Ca +/calmodulin-dependent protein kinase II and cAMP-dependent protein kinase which suggests a role for both intracellular Ca + and enzyme phosphorylation in the activation of tryptophan hydroxylase. Indeed, enzyme purified from brain tissue innervated by rostrally projecting 5-HT neurons, that have been stimulated previously in vivo, has a higher activity than that derived from unstimulated tissue but this increase rests on the presence of Ca + in the incubation medium. Also, when incubated under conditions which are appropriate for phosphorylation, the of tryptophan hydroxylase for its co-factor and substrate is reduced whereas its Fmax is increased unless the enzyme is purified from neurons that have been stimulated in vivo, suggesting that the neuronal depolarisation in vivo has already caused phosphorylation of the enzyme. This is supported by evidence that the enzyme activation caused by neuronal depolarisation is blocked by a Ca +/calmodulin protein kinase inhibitor. However, whereas depolarisation... 

These data demonstrate that both GSH and GSSG have profound effects on Na/K ATPase activity and may act in concert to modify enzyme activity during oxidant stress. However, it should be recognized that the steric conformation of an isolated enzyme preparation in a chemically buffered solution may be considerably different to the native enzyme located in a dynamic lipid bilayer. For this reason, these investigations have been extended to include a variety of preparations in which the Na/K pump is in its native environment. 

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