Triosephosphate isomerase, catalytic activity

Less dramatic conformational changes are involved in cleft closure or closure of lids over active sites in many enzymes, including triosephosphate isomerase, hexokinase, phosphonatase, and adenylosuccinate lyase." Such motions sequester active sites, preventing escape of reactive intermediates or inadvertent reaction with the solvent, and often help to orient substrates and/or catalytic residues. 

The fecat/J M ratios of the enzymes superoxide dismutase, acetylcholinesterase, and triosephosphate isomerase are between 10 and lO" s Enzymes such as these that have fecat/ M ratios at the upper limits have attained kinetic perfection. Their catalytic velocity is restricted only by the rate at which they encounter substrate in the solution (Table 8.8). Any further gain in catalytic rate can come only by decreasing the time for diffusion. Remember that the active site is only a small part of the total enzyme structure. Yet, for catalytically perfect enzymes, every encounter between enzyme and substrate is productive. In these cases, there may be attractive electrostatic forces on the enzyme that entice the substrate to the active site. These forces are sometimes referred to poetically as Circe effects. 

The most successful application of haloacetol phosphates as affinity labels has been in the partial characterization of the active site of triosephosphate isomerase. Very similar studies, carried out independently in the laboratories of F. C. Hartman and J. R. Knowles, demonstrated an essential glutamyl y-carboxylate (esterified by the reagent) in the enzyme. The recently determined primary structure of the enzyme from rabbit muscle places the glutamyl at position 165. All the usual criteria of affinity labeling were satisfied and have been well documented. Certain aspects of these studies that either relate to the use of haloketones in general or have provided evidence of the carboxylate s intimate role in the catalytic process will be considered. 

Studies with ab initio types of hybrid potential include the early work of Weiner et al. on the nature of catalysis in trypsin and the studies of the catalytic activity of phospholipase A2 by Hillier et al. Investigations with semiempirical hybrid potentials are more extensive and include calculations of the reactions in triosephosphate isomerase by Bash et al. and in chorismate mutase by Lyne et al. and a study of the proton jump in the catalytic triad of human neutrophil elastase. The study of the chorismate mutase reaction was especially interesting because the enzyme is the only known one that catalyzes a pericyclic reaction that also occurs readily in solution. The results of the hybrid study were particularly lucid in this case because the enzyme works, not by chemically catalyzing the reaction, but by preferentially binding a distorted form of the substrate and stabilizing the transition state. 

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