Amino acid composition and effect of pH on the kinetic parameters of renal dipeptidase.

The procedure for the purification of renal dipeptidase from particulate kidney cortex fractions has been modified to eliminate a time-consuming washing process and a chromatographic step which frequently led to loss in activity. The over-all purification resulted in the isolation of 5.8 mg of purified peptidase from 1.5 kg of kidney cortex and produced an enzyme approximately 500 times more active than source material. Physical and chemical properties such as sedimentation characteristics, electrophoretic behavior, and zinc content were compared with those reported for earlier peptidase preparations. The amino acid composition of the enzyme based on a molecular weight of 47,200 indicates a protein that contains approximately 414 amino acid residues. A study of the effect of pH upon the kinetic parameters of renal dipeptidase indicates that the NHz-terminal amino group of the peptide substrate dissociates a proton during the catalytic process. Furthermore, it is suggested that a dissociation of pK 8.5 which takes place from the enzymesubstrate complex results from the loss of a proton from a water molecule coordinated to zinc at the active center of the enzyme.