Expression and Characterization of Human Pancreatic Preprocarboxypeptidase A1 and Preprocarboxypeptidase A2

Abstract

We are investigating the potential utility of human carboxypeptidases A in antibody-directed enzyme prodrug therapy (ADEPT). Hybridization screening of a human pancreatic cDNA library with cDNA probes that encoded either rat carboxypeptidase A1 (rCPA1) or carboxypeptidase A2 (rCPA2) was used to clone the human prepro-CPA homologs. After expression of the respective pro-hCPA cDNA inSaccharomyces cerevisiae,the enzymes were purified to homogeneity by a combination of hydrophobic and ion-exchange chromatography. Purified hCPA1 and hCPA2 migrate as a single protein band withMr34,000 when subjected to gel electrophoresis in the presence of sodium dodecyl sulfate under reducing conditions. Kinetic studies of the purified enzymes with hippuryl-L-phenylalanine resulted inkcat/Kmvalues of 57,000 and 19,000M−1s−1for hCPA1 and hCPA2, respectively. Using the ester substrate, hippuryl-D,L-phenyllactate, we found unique esterase/peptidase specific activity ratios among hCPA1, hCPA2, rCPA1, and bovine CPA (bCPA) ranging from 13 to 325. Two potential ADEPT substrates, methotrexate–α-phenylalanine (MTX-Phe) and methotrexate–α-(1-naphthyl)alanine (MTX-naphthylAla) were also analyzed. Thekcat/Kmvalues for MTX-Phe were 440,000 and 90,000M−1s−1for hCPA1 and hCPA2, respectively, and for MTX-naphthylAla these values were 1400 and 1,400,000M−1s−1for hCPA1 and hCPA2, respectively. The kinetic data show that hCPA2 has a larger substrate binding site than the hCPA1 enzyme. Differences between hCPA1 and hCPA2 were also observed in thermal stability experiments at 60°C where the half-life for thermal denaturation of hCPA2 is eightfold longer than that for hCPA1. These experiments indicate that hCPA1 and hCPA2 are potential candidates for use in a human-based ADEPT approach.