Kinetic and Spectroscopic Analysis of the Catalytic Role of H79 in the Methionine Aminopeptidase from Escherichia coli

Authors

Sarah J. Watterson, Utah State University
Sanghamitra Mitra, Utah State UniversityFollow
Sabina I. Swierczek, Utah State UniversityFollow
Brian Bennett, Marquette UniversityFollow
Richard C. Holz, Marquette UniversityFollow

Document Type

Article

Language

eng

Format of Original

9 p.

Publication Date

11-2008

Publisher

American Chemical Society

Source Publication

Biochemistry

Source ISSN

0006-2960

Original Item ID

doi: 10.1021/bi801499g

Abstract

To gain insight into the role of the strictly conserved histidine residue, H79, in the reaction mechanism of the methionyl aminopeptidase from Escherichia coli (EcMetAP-I), the H79A mutated enzyme was prepared. Co(II)-loaded H79A exhibits an overall >7000-fold decrease in specific activity. The almost complete loss of activity is primarily due to a >6000-fold decrease in k_cat. Interestingly, the K_m value obtained for Co(II)-loaded H79A was approximately half the value observed for wild-type (WT) EcMetAP-I. Consequently, k_cat/K_m values decreased only 3000-fold. On the other hand, the observed specific activity of Mn(II)-loaded H79A EcMetAP-I decreased by ∼2.6-fold while k_cat decreased by ∼3.5-fold. The observed K_m value for Mn(II)-loaded H79A EcMetAP-I was ∼1.4-fold larger than that observed for WT EcMetAP-I, resulting in a k_cat/K_m value that is lower by ∼3.4-fold. Metal binding, UV−vis, and EPR data indicate that the active site is unperturbed by mutation of H79, as suggested by X-ray crystallographic data. Kinetic isotope data indicate that H79 does not transfer a proton to the newly forming amine since a single proton is transferred in the transition state for both the WT and H79A EcMetAP-I enzymes. Therefore, H79 functions to position the substrate by hydrogen bonding to either the amine group of the peptide linkage or a backbone carbonyl group. Together, these data provide new insight into the catalytic mechanism of EcMetAP-I.

Comments

Accepted version. Biochemistry, Vol. 47, No. 45 (November 2008): 11885–11893. DOI. © 2008 American Chemical Society Publications. Used with permission.

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

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

Recommended Citation

Watterson, Sarah J.; Mitra, Sanghamitra; Swierczek, Sabina I.; Bennett, Brian; and Holz, Richard C., "Kinetic and Spectroscopic Analysis of the Catalytic Role of H79 in the Methionine Aminopeptidase from Escherichia coli" (2008). Physics Faculty Research and Publications. 12.
https://epublications.marquette.edu/physics_fac/12