The objective of this work is to characterize in mechanistic detail the way by which certain hydrolytic enzymes function. Information obtained from these studies could contribute to the development of improved methods for treating and characterizing certain metabolic diseases in man which arise from an abnormality in a hydrolytic enzyme. Interactive effects of ionizable groups at the active site of papain on the ionization of the active site thiol group will be investigated by pH-difference titrations of derivatives of papain relative to papain. The ionization of the two histidyl residues of papain will be studied by means of proton-nmr titrations. The mobility and accessibility of the active site thiol group in papain will be examined by halide ion probe-nmr. The environment at the active site of papain will be investigated with reporter groups linked to the active site thiol group. Individual steps in the catalytic pathway will be examined using C13-enriched dinitrophenyl peptides by means of C13-nmr and stopped-flow spectrometry. We will also consider the effect of the activator proflavine on individuals steps in the catalytic pathway of papain. Interactions of derivatives of D-serine with D-serine dehydratase will be studied, in order to characterize the way this enzyme interacts with its substrates. Individual steps in the catalytic pathway for the D-serine dehydratase-catalyzed reaction will be characterized by studying the presteady-state, and kinetic isotope effects on the presteady-state. The lactonization of benzamide derivatives containing neighboring thiol and hydroxyl groups will be examined as a model for the action of certain hydrolytic enzymes.