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

Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322-0300, USA.
Biochemistry (Impact Factor: 3.02). 10/2008; 47(45):11885-93. DOI: 10.1021/bi801499g
Source: PubMed


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.

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Available from: Brian Bennett, Feb 06, 2014
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