Kinetic and spectroscopic analysis of the catalytic role of H79 in the methionine aminopeptidase from Escherichia coli
Graduate School of Biomedical Sciences; Department of Biochemistry and Molecular Pharmacology
Life Sciences | Medicine and Health Sciences
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.
DOI of Published Version
Biochemistry. 2008 Nov 11;47(45):11885-93. Epub 2008 Oct 15. Link to article on publisher's site
Watterson, Sarah J.; Mitra, Sanghamitra; Swierczek, Sabina I.; Bennett, Brian Thomas; and Holz, Richard C., "Kinetic and spectroscopic analysis of the catalytic role of H79 in the methionine aminopeptidase from Escherichia coli" (2008). GSBS Student Publications. 1404.