Mutation of H63 and Its Catalytic Affect on the Methionine Aminopeptidase from Escherichia coli

Authors

Sanghamitra Mitra, Boston UniversityFollow
Brian Bennett, Marquette UniversityFollow
Richard C. Holz, Marquette UniversityFollow

Document Type

Article

Language

eng

Format of Original

7 p.

Publication Date

1-2009

Publisher

Elsevier

Source Publication

Biochimica et Biophysica Acta: Proteins and Proteomics

Source ISSN

0006-3002

Original Item ID

doi: 10.1016/j.bbapap.2008.09.012

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.

Comments

Accepted version. Biochimica et Biophysica Acta: Proteins and Proteomics, Vol.1794, No.1 (January 2009): 137-143. DOI. © 2009 Elsevier B.V. Used with permission.

Brian Bennett was affiliated with Medical College of Wisconsin at the time of publication.

Richard Holz was affiliated with Loyola University-Chicago at the time of publication.

Recommended Citation

Mitra, Sanghamitra; Bennett, Brian; and Holz, Richard C., "Mutation of H63 and Its Catalytic Affect on the Methionine Aminopeptidase from Escherichia coli" (2009). Physics Faculty Research and Publications. 85.
https://epublications.marquette.edu/physics_fac/85