Comparison of Substrate Specificity of Escherichia Coli p -Aminobenzoyl-Glutamate Hydrolase with Pseudomonas Carboxypeptidase G

• Published in: Advances in enzyme research Vol. 2; no. 1; pp. 39 - 48
• Main Authors: Larimer, Cassandra M, Slavnic, Dejan, Pitstick, Lenore D, Green, Jacalyn M
• Format: Journal Article
• Language: English
• Published: United States 01.03.2014
• Subjects: Folate Catabolism; p-Aminobenzoyl-Glutamate Hydrolase; Carboxypeptidase G
• ISSN: 2328-4846; 2328-4854
• DOI: 10.4236/aer.2014.21004

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 is -aminobenzoyl-glutamate (PABA-GLU). The enzyme -aminobenzoyl-glutamate hydrolase (PGH) breaks down PABA-GLU and is part of an apparent operon, the region, in . The subunits of PGH possess sequence and catalytic similarities to carboxypeptidase enzymes from species. 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 G from RS-16, which has been crystallized. Then we compared the enzyme activities of PGH and commercially available carboxypeptidase G using spectrophotometric assays measuring cleavage of PABA-GLU, folate, aminopterin, methotrexate, 5-formyltetrahydrofolate, and 5-methyltetrahydrofolate. The and values 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 that PGH 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.