EXPRESSION AND CHARACTERIZATION OF HUMAN PANCREATIC PREPROCARBOXYPEPTIDASE A1 AND PREPROCARBOXYPEPTIDASE A2

We are investigating the potential utility of human carboxypeptidases A in antibody-directed enzyme prodrug therapy (ADEPT). Hybridization s creening of a human pancreatic cDNA library with cDNA probes that enco ded either rat carboxypeptidase A1 (rCPA1) or carboxypeptidase A2 (rCP A2) was used to clone the human prepro-CPA homologs. After expression of the respective pro-hCPA cDNA in Saccharomyces cerevisiae, the enzym es were purified to homogeneity by a combination of hydrophobic and io n-exchange chromatography. Purified hCPA1 and hCPA2 migrate as a singl e protein band with M(r) 34,000 when subjected to gel electrophoresis in the presence of sodium dodecyl sulfate under reducing conditions. K inetic studies of the purified enzymes with hippuryl-L-phenylalanine r esulted in k(cat)/K-m values of 57,000 and 19,000 M(-1) s(-1) for hCPA 1 and hCPA2, respectively. Using the ester substrate, hippuryl-D,L-phe nyllactate, we found unique esterase/peptidase specific activity ratio s among hCPA1, hCPA2, rCPA1, and bovine CPA (bCPA) ranging from 13 to 325. Two potential ADEPT substrates, methotrexate-alpha-phenylalanine (MTX-Phe) and methotrexate-alpha-(1-naphthyl)alanine (MTX-naphthylAla) were also analyzed. The k(cat)/K-m values for MTX-Phe were 440,000 an d 90,000 M(-1) s(-1) for hCPA1 and hCPA2, respectively, and for MTX-na phthylAla these values were 1400 and 1,400,000 M(-1) s(-1) for hCPA1 a nd hCPA2, respectively. The kinetic data show that hCPA2 has a larger substrate binding site than the hCPA1 enzyme. Differences between hCPA 1 and hCPA2 were also observed in thermal stability experiments at 60 degrees C where the half-life for thermal denaturation of hCPA2 is eig htfold longer than that for hCPA1. These experiments indicate that hCP A1 and hCPA2 are potential candidates for use in a human-based ADEPT a pproach. (C) 1996 Academic Press, Inc.