Cloning, Overexpression, and Crystallization of Proline Racemase

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9407346 Allen This is a Research Planning Grant. The proposed research to be conducted during the planning period is the cloning, overexpression and crystallization of the enzyme proline racemase. The research following the proposed planning period is the determination of the structure of proline racemase by X-ray crystallography in the native state and in the presence of substrates, substrate analogs and transition state analogs. Proline racemase has been shown to induce strain/destabilization of substrate, one of four mechanisms by which enzymes bring about rate accelarations. The induced strain is relieved in the enzyme-transition state complex. Of the modes of catalysis, the least understood is strain/destabilization since it is difficult to devise experiments for its evaluation. The comparison of the X-ray crystal structures of the enzyme-substrate and enzyme- transition state analog complexes of proline racemase will allow direct observation of the structural and chemical means by which this enzyme achieves strain/destabilization of the substrate. %%% There are few cases in which enzymes have been proven to undergo strain/destabilization in the enzyme-substrate complex. Proline racemase is the sole example in which transition state and substrate analogs exist and which is amenable to study by X-ray crystallography. The structure of these complexes will provide the basis by which we can understand the strain/destabilization mode of catalysis. ***