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

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9 p.

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American Chemical Society

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doi: 10.1021/bi801499g


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 kcat. Interestingly, the Km value obtained for Co(II)-loaded H79A was approximately half the value observed for wild-type (WT) EcMetAP-I. Consequently, kcat/Km 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 kcat decreased by ∼3.5-fold. The observed Km value for Mn(II)-loaded H79A EcMetAP-I was ∼1.4-fold larger than that observed for WT EcMetAP-I, resulting in a kcat/Km 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.


Biochemistry, Vol. 47, No. 45 (November 2008): 11885–11893. DOI.

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.