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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.


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.

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