title: Comparison of Substrate Specificity ofEscherichia Coli p-Aminobenzoyl-Glutamate Hydrolase withPseudomonasCarboxypeptidase G.
abstract: Reduced folic acid derivatives support biosynthesis of DNA, RNA and amino acids in bacteria as well as in eukaryotes, including humans. While the genes and steps for bacterial folic acid synthesis are known, those associated with folic acid catabolism are not well understood. A folate catabolite found in both humans and bacteria isp-aminobenzoyl-glutamate (PABA-GLU). The enzymep-aminobenzoyl-glutamate hydrolase (PGH) breaks down PABA-GLU and is part of an apparent operon, theabgregion, inE. coli. The subunits of PGH possess sequence and catalytic similarities to carboxypeptidase enzymes fromPseudomonasspecies. A comparison of the subunit sequences and activity of PGH, relative to carboxypeptidase enzymes, may lead to a better understanding of bacterial physiology and pathway evolution. We first compared the amino acid sequences of AbgA, AbgB and carboxypeptidase G2fromPseudomonas sp.RS-16, which has been crystallized. Then we compared the enzyme activities ofE. coliPGH and commercially availablePseudomonascarboxypeptidase G using spectrophotometric assays measuring cleavage of PABA-GLU, folate, aminopterin, methotrexate, 5-formyltetrahydrofolate, and 5-methyltetrahydrofolate. TheKmandVmaxvalues for the folate and anti-folate substrates of PGH could not be determined, because the instrument reached its limit before the enzyme was saturated. Therefore, activity of PGH was compared to the activity of CPG, or normalized to PABA-GLU (nmole/min/µg). Relative to its activity with 10 µM PABA-GLU (100%), PGH cleaved glutamate from methotrexate (48%), aminopterin (45%) and folate (9%). Reduced folates leucovorin (5-formyltetrahydrofolate) and 5-methyltetrahydrofolate were not cleaved by PGH. Our data suggest thatE. coliPGH is specific for PABA-GLU as its activity with natural folates (folate, 5-methyltetrahydrofolate, and leucovorin) was very poor. It does, however, have some ability to cleave anti-folates which may have clinical applications in treatment of chemotherapy overdose.
authors: Department of Biochemistry, Chicago College of Osteopathic Medicine, Midwestern University, Downers Grove, IL, USA.
authors: Cassandra M
authors: CM
authors: Larimer
authors: Department of Biochemistry, Chicago College of Osteopathic Medicine, Midwestern University, Downers Grove, IL, USA.
authors: Dejan
authors: D
authors: Slavnic
authors: Department of Biochemistry, Chicago College of Osteopathic Medicine, Midwestern University, Downers Grove, IL, USA.
authors: Lenore D
authors: LD
authors: Pitstick
authors: Department of Biochemistry, Chicago College of Osteopathic Medicine, Midwestern University, Downers Grove, IL, USA.
authors: Jacalyn M
authors: JM
authors: Green
keywords: Carboxypeptidase G; Folate Catabolism; p-Aminobenzoyl-Glutamate Hydrolase