Purification and Properties of Carboxypeptidase G1

An enzyme, carboxypeptidase G1, extracted from a strain of Pseudomonas stutzeri, has been isolated which hydrolyzes the carboxyl-terminal glutamate from both reduced and nonreduced folate derivatives, with Km values between 1.1 and 18.1 x 10-6 m. This enzyme also hydrolyzes the COOH-terminal glutamate of oligopeptides and N-benzyloxycarbonyl glutamates; it demonstrates lesser activity against aspartate COOH-terminal peptide linkages, with Km values of 1 to 5 x 10-4 m. The reverse reaction (pteroate + glutamate → folate) is also catalyzed, but at an equilibrium lying far toward hydrolysis. The enzyme has been purified 1050-fold. It has a broad pH optimum over the range of 6.3 to 7.3 and has a molecular weight of 92,000 as estimated by calibrated Sephadex gel filtration. On disc gel electrophoresis in 1% sodium dodecyl sulfate, purified preparations migrate as a single band of 46,000 molecular weight, indicating that the enzyme is a dimeric protein. The enzyme is activated by Zn++. Enzymic cleavage of 4-amino-N10-methylpteroylglutamic acid (methotrexate) was strongly inhibited by the product l-glutamic acid, and to a lesser extent by l-aspartic acid, while d-glutamic acid was ineffective. Competitive inhibition of methotrexate cleavage was also demonstrated by the substrates 4-aminopteroylaspartic acid, N-benzyloxycarbonyl glutamic acid, and N-benzyloxycarbonyl aspartic acid. Although certain properties of carboxypeptidase G1 were similar to those from other sources, several differences were noted between this enzyme and previously described carboxypeptidases.