GSBS Student Publications
Title
Mutation of H63 and its catalytic affect on the methionine aminopeptidase from Escherichia coli
Publication Date
2008-10-28
UMMS Affiliation
Graduate School of Biomedical Sciences; Department of Biochemistry and Molecular Pharmacology
Document Type
Article
Disciplines
Life Sciences | Medicine and Health Sciences
Abstract
In order to gain insight into the mechanistic role of a flexible exterior loop near the active site, made up of Y62, H63, G64, and Y65, that has been proposed to play an important role in substrate binding and recognition in the methionyl aminopeptidase from Escherichia coli (EcMetAP-I), the H63A enzyme was prepared. Mutation of H63 to alanine does not affect the ability of the enzyme to bind divalent metal ions. The specific activity of H63A EcMetAP-I was determined using four different substrates of varying lengths, namely, l-Met-p-NA, MAS, MGMM and MSSHRWDW. For the smallest/shortest substrate (l-Met-p-NA) the specific activity decreased nearly seven fold but as the peptide length increased, the specific activity also increased and became comparable to WT EcMetAP-I. This decrease in specific activity is primarily due to a decrease in the observed k(cat) values, which decreases nearly sixty-fold for l-Met-p-NA while only a four-fold decrease is observed for the tri- and tetra-peptide substrates. Interestingly, no change in k(cat) was observed when the octa-peptide MSSHRWDW was used as a substrate. These data suggest that H63 affects the hydrolysis of small peptide substrates whereas large peptides can overcome the observed loss in binding energy, as predicted from K(m) values, by additional hydrophilic and hydrophobic interactions.
DOI of Published Version
10.1016/j.bbapap.2008.09.012
Source
Biochim Biophys Acta. 2009 Jan;1794(1):137-43. Epub 2008 Oct 7. Link to article on publisher's site
Journal/Book/Conference Title
Biochimica et biophysica acta
Related Resources
PubMed ID
18952013
Repository Citation
Mitra, Sanghamitra; Bennett, Brian Thomas; and Holz, Richard C., "Mutation of H63 and its catalytic affect on the methionine aminopeptidase from Escherichia coli" (2008). GSBS Student Publications. 1409.
https://escholarship.umassmed.edu/gsbs_sp/1409