Comparison of Substrate Specificity of Escherichia Coli p-Aminobenzoyl-Glutamate Hy-drolase with Pseudomonas Carboxypeptidase GReportar como inadecuado




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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 p-aminobenzoyl-glutamate PABA-GLU. The enzyme p-aminobenzoyl-glutamate hydrolase PGH breaks down PABA-GLU and is part of an apparent operon, the abg region, in E. coli. The subunits of PGH possess sequence and catalytic similarities to carboxypeptidase enzymes from Pseudomonas 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 G2 from Pseudomonas sp. RS-16, which has been crystallized. Then we compared the enzyme activities of E. coli PGH and commercially available Pseudomonas carboxypeptidase G using spectrophotometric assays measuring cleavage of PABA-GLU, folate, aminopterin, methotrexate, 5-formyltetrahydrofolate, and 5-methyltetrahydrofolate. The Km and Vmax 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 E. coli 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.

KEYWORDS

Folate; p-Aminobenzoyl-Glutamate Hydrolase; Carboxypeptidase G; Folate Catabolism

Cite this paper

Larimer, C. , Slavnic, D. , Pitstick, L. and Green, J. 2014 Comparison of Substrate Specificity of Escherichia Coli p-Aminobenzoyl-Glutamate Hy-drolase with Pseudomonas Carboxypeptidase G. Advances in Enzyme Research, 2, 39-48. doi: 10.4236-aer.2014.21004.





Autor: Cassandra M. Larimer, Dejan Slavnic, Lenore D. Pitstick, Jacalyn M. Green

Fuente: http://www.scirp.org/



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