Medical College of Wisconsin
CTSICores SearchResearch InformaticsREDCap

Kinetic and spectroscopic analysis of the catalytic role of H79 in the methionine aminopeptidase from Escherichia coli. Biochemistry 2008 Nov 11;47(45):11885-93

Date

10/16/2008

Pubmed ID

18855426

Pubmed Central ID

PMC2720795

DOI

10.1021/bi801499g

Scopus ID

2-s2.0-55849098590 (requires institutional sign-in at Scopus site)   11 Citations

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 approximately 2.6-fold while k cat decreased by approximately 3.5-fold. The observed K m value for Mn(II)-loaded H79A EcMetAP-I was approximately 1.4-fold larger than that observed for WT EcMetAP-I, resulting in a k cat/ K m value that is lower by approximately 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.

Author List

Watterson SJ, Mitra S, Swierczek SI, Bennett B, Holz RC

Author

Brian Bennett D.Phil. Professor and Chair in the Physics department at Marquette University




MESH terms used to index this publication - Major topics in bold

Aminopeptidases
Binding Sites
Catalysis
Cobalt
Crystallography, X-Ray
Electron Spin Resonance Spectroscopy
Escherichia coli
Escherichia coli Proteins
Histidine
Iron
Kinetics
Methionyl Aminopeptidases
Models, Molecular
Mutagenesis, Site-Directed