Papain, a thiol protease from papaya latex, will be studied to determine the role of Asp-158 in catalysis and to determine the effect of Asp-158 on the interactive ionization of His-159 and Cys-25 at the active site of papain. Derivatives of papain modified at Asp-158 will be prepared and characterized with respect to their altered interactions with substrates and inhibitors. Proton NMR and potentiometric difference titrations will be used to determine alterations in the ionization behavior of His-159 and Cys-25 caused by modification of Asp-158. The interactive ionization of His-159 and Cys-25 also will be investigated to determine the effect of the ion-pair interaction on the reactivity of these residues. The nucleophilic reactivity of ammonium-thiolate ion-pairs in nonenzymic reactions will be studied to evaluate the possible catalytic advantage of the imidazolium-thiolate ion-pair at the active site of papain. The potentiometric difference titration method we have developed for determining the ionization behavior of the thiol group in papain will be used to determine the possible existence of ligand dependent ionic interactions involving Cys-beta 93 of hemoglobin. D-Serine dehydratase from E. coli will be studied to determine a) how monovalent cations effect the affinity of the enzyme for its cofactor pyridoxal 5'-phosphate, b) the involvement of a thiol group in the catalytic activity of the enzyme, and c) the intermediates in the catalytic pathway and their rates of interconversion. Studies with human fibrinogen from individuals with dysfibrinogenemia are proposed in which amino acid replacements in abnormal fibrinogens will be related to their altered functional competence especially their altered interactions with the enzymes involved in blood clot formation dissolution.
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