title: 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 in Saccharomyces 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 with M(r) 34,000 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 in kcat/Km values of 57,000 and 19,000 M-1 s-1 for 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-alpha-phenylalanine (MTX-Phe) and methotrexate-alpha-(1-naphthyl)alanine (MTX-naphthylAla) were also analyzed. The kcat/Km values for MTX-Phe were 440,000 and 90,000 M-1 s-1 for hCPA1 and hCPA2, respectively, and for MTX-naphthylAla these values were 1400 and 1,400,000 M-1 s-1 for 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 degrees 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.
authors.affiliation: Division of Cell Biology, Wellcome Research Laboratories, Research Triangle Park, North Carolina 27709, USA.
authors.forename: R M
authors.initials: RM
authors.lastname: Laethem
authors.forename: T A
authors.initials: TA
authors.lastname: Blumenkopf
authors.forename: M
authors.initials: M
authors.lastname: Cory
authors.forename: L
authors.initials: L
authors.lastname: Elwell
authors.forename: C P
authors.initials: CP
authors.lastname: Moxham
authors.forename: P H
authors.initials: PH
authors.lastname: Ray
authors.forename: L M
authors.initials: LM
authors.lastname: Walton
authors.forename: G K
authors.initials: GK
authors.lastname: Smith
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002268:Carboxypeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002417:Cattle; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004792:Enzyme Precursors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004795:Enzyme Stability; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D008727:Methotrexate / Q000031:analogs & derivatives / Q000378:metabolism / Q000627:therapeutic use; D008969:Molecular Sequence Data; D009369:Neoplasms / Q000188:drug therapy; D010179:Pancreas / Q000201:enzymology*; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000378:metabolism / Q000627:therapeutic use; D011355:Prodrugs / Q000378:metabolism / Q000627:therapeutic use; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D017931:DNA Primers; D004792:Enzyme Precursors; D011355:Prodrugs; C091716:methotrexate-alpha-phenylalanine; D010649:Phenylalanine; C049123:N-(hippuryl)phenylalanine; D002268:Carboxypeptidases; C033512:preprocarboxypeptidase A; D008727:Methotrexate