Carboxypeptidase displaying differential velocity in hydrolysis of methotrexate, 5-methyltetrahydrofolic acid, and leucovorin

Abstract

An enzyme that catalyzes the hydrolysis of folic acid and the antifolate methotrexate nearly 20 times more rapidly than the hydrolysis of 5-methyltetrahydrofolate was extracted from a gram-negative bacterium tentatively identified as a Flavobacterium sp. The enzyme was purified 500-fold and found to have a molecular weight of about 53,000. Apparently a metallo-enzyme, it is inhibited by citrate and ethylenediaminetetraacetic acid (EDTA). Ca2+, Co2+, Mg2+, and Zn2+ reverse inhibition by EDTA, whereas Ca2+ and Zn2+ are weak activators in the absence of EDTA. The enzymatic reaction releases the carboxy-terminal glutamyl moiety of derivatives of pteroyl-mono-L-glutamic acid. Substituents on N5 of the pteridine ring decrease the velocity of hydrolysis. Some non-specificity for the terminal amino acid is expressed. The strikingly different rates of hydrolysis of methotrexate and 5-methyltetrahydrofolate have stimulated interest in this enzyme for its potential clinical value in improving the therapeutic index of methotrexate. The folic acid analog methotrexate (4-amino-N10-methylpteroylglutamic acid [MTX]) is a potent antineoplastic agent (1). Its clinical application has been advanced with the use of leucovorn (N6-formyltetrahydrofolic acid) rescue (8, 11, 13, 18, 26), which has been relatively successful in relieving the toxicity of antifolate therapy. The report of a bacterial carboxypeptidase G1 (CPD G1) with hydrolytic activity for MTX introduced the possibility of the enzymatic approach to ameliorating drug toxicity (12, 23). In this report, we describe the partial purification and characterization of another enzyme that catalyzes the hydrolysis of MTX, folic acid (FA), and other derivatives of FA by the reaction shown in Fig. 1. This carboxypeptidase is produced by a bacterium tentatively designated Flavobacterium sp. and differs from CPD G and CPD G1 (15, 19, 23). MATERIALS AND METHODS Chemicals. FA (pteroyl-L-glutamic acid) was purchased from Grand Island Biological Co. dl-N5-methyltetrahydrofolic acid was obtained from Sigma Chemical Co. as the barium salt. It was converted to the sodium salt by the addition of an equal volume of 0.1 M sodium phosphate, pH 6.5, to a 1% solution of the folate in 0.1 M sodium bicarbonate-0.05% potassium ascorbate. Leucovorin (dl-N-5-formyltetrahydrofolic acid, calcium salt) and MTX were provided by Bernard C. Clark of Lederle Laboratories, American Cy-anamid Co., and by Harry B. Wood, Jr., Drug Development Branch, Drug Research and Development, Silver Spring, Md. Chromatographicaily purified MTX (4) was used for kinetic studies. Purified aminopterin was a gift from Francis M. Sirotnak, Sloan-Kettering Institute. Harry B. Wood, Jr., also provided bioautographically pure samples of methasquin (17),

Understanding and Managing Methotrexate Nephrotoxicity

“…The carboxypeptidase-G class of enzymes hydrolyze the terminal glutamate from naturally occurring folates and folate analogs, such as MTX [60]. Carboxypeptidase-G rapidly converts MTX to the inactive metabolites DAMPA and glutamic acid, thus providing an alternate route of elimination to renal excretion.…”

Characterization of the folate salvage enzyme p-aminobenzoylglutamate hydrolase in plants

“…Nor, with one exception, is much known about pABAGlu-hydrolyzing enzymes from other organisms. The only well-characterized protein known to hydrolyze pABAGlu 5 is carboxypeptidase G (CPG, EC 3.4.17.11), a di-zinc enzyme from Pseudomonas and other bacteria (McCullough et al, 1971;Albrecht et al, 1978;Sherwood et al, 1985). CPG also cleaves the pABA-glutamate bond in folates and folate analogs, releasing pteroate 3 and glutamate 6 fragments, and can remove the γ-glutamyl tail from polyglutamates by exopeptidase action.…”

Efficacy of Glucarpidase (Carboxypeptidase G2) in Patients with Acute Kidney Injury After High‐Dose Methotrexate Therapy

“…3,15 High-flux hemodialysis is the most effective method of extracorporeal methotrexate removal but requires five to six daily treatments (4-6 hours per session) with a very high blood flow rate (400 ml/minute) 16 ; it often results in transient rebound increases in methotrexate concentrations and has been associated with low blood pressure, fatigue, chest pain, leg cramps, nausea, and headaches. [17][18][19] Glucarpidase, previously known as carboxypeptidase G2, is a carboxypeptidase G enzyme isolated from Pseudomonas species strain RS-16 that hydrolyzes the terminal glutamate residue from methotrexate, along with other naturally occurring folates and folate analogs, 20 and it was recently approved by the United States Food and Drug Administration (Voraxaze; BTG International, Inc., West Conshohocken, PA). The hydrolysis of methotrexate and its active metabolite 7-hydroxymethotrexate by glucarpidase forms the inactive metabolites glutamate, 2,4-diamino-N 10methylpteroic acid (DAMPA), 21 and (OH)-DAMPA, which are partially metabolized by the liver.…”

“…Glucarpidase, previously known as carboxypeptidase G2, is a carboxypeptidase G enzyme isolated from Pseudomonas species strain RS‐16 that hydrolyzes the terminal glutamate residue from methotrexate, along with other naturally occurring folates and folate analogs, and it was recently approved by the United States Food and Drug Administration (Voraxaze; BTG International, Inc., West Conshohocken, PA). The hydrolysis of methotrexate and its active metabolite 7‐hydroxymethotrexate by glucarpidase forms the inactive metabolites glutamate, 2,4‐diamino‐N 10 ‐methylpteroic acid (DAMPA), and (OH)‐DAMPA, which are partially metabolized by the liver .…”