Utilization of conformational flexibility in enzyme action-linkage between binding, isomerization, and catalysis.

An intimate relationship between protein conformational changes and catalysis has often been suggested. The present study employs ligand-induced ultraviolet difference spectra and kinetic parameters determined for Escherichia coli ornithine transcarbamoylase and its site-specific mutants to evaluate the linkage between binding, isomerization, and reaction rate. For the wild-type enzyme, the lead substrate carbamoyl phosphate introduces a large difference absorbance in the enzyme upon binding (delta epsilon max approximately 1,800 M-1 cm-1; Miller, A. W., and Kuo, L. C. (1990) J. Biol. Chem. 265, 15023-15027). The spectrum is the same in lineshape as that produced by the bisubstrate analog N-(phosphonacetyl)-L-ornithine and is 80% as intense. Both substrate and analog cause gross protein conformational rearrangements as evident by swift and severe cracking of enzyme crystals in their presence. For the mutants, the difference spectra actuated by the substrate are the same in lineshape as that of the wild type but vary in intensity. A wide range of substrate affinity and steady-state kinetic constants are also observed for the mutants. When the binding energy of carbamoyl phosphate and the activation energy for transcarbamoylation are calculated for the wild-type and mutant enzymes, they are found to be inversely correlated to the intensity of protein difference absorbance elicited by the lead substrate. Together with analyses of steady-state kinetic parameters derived for various plausible reaction schemes, the experimental data suggest that carbamoyl phosphate induces the committed isomerization in ornithine transcarbamoylase for transition state binding. Our results provide a unique demonstration that an induced-fit isomerization, triggered by binding, either controls or contributes significantly to the rate of an enzyme-catalyzed reaction.