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Biochemistry. 2003 May 27;42(20):6283-92.

Kinetic and spectroscopic characterization of the H178A methionyl aminopeptidase from Escherichia coli.

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Department of Chemistry and Biochemistry, Utah State University, Logan 84322-0300, USA.


To gain insight into the role of the strictly conserved histidine residue, H178, in the reaction mechanism of the methionyl aminopeptidase from Escherichia coli (EcMetAP-I), the H178A mutant enzyme was prepared. Metal-reconstituted H178A binds only one equivalent of Co(II) or Fe(II) tightly with affinities that are identical to the WT enzyme based on kinetic and isothermal titration calorimetry (ITC) data. Electronic absorption spectra of Co(II)-loaded H178A EcMetAP-I indicate that the active site divalent metal ion is pentacoordinate, identical to the WT enzyme. These data indicate that the metal binding site has not been affected by altering H178. The effect of altering H178 on activity is, in general, due to a decrease in k(cat). The k(cat) value for Co(II)-loaded H178A decreased 70-fold toward MGMM and 290-fold toward MP-p-NA compared to the WT enzyme, while k(cat) decreased 50-fold toward MGMM for the Fe(II)-loaded H178A enzyme and 140-fold toward MP-p-NA. The K(m) values for MGMM remained unaffected, while those for MP-p-NA increased approximately 2-fold for Co(II)- and Fe(II)-loaded H178A. The k(cat)/K(m) values for both Co(II)- and Fe(II)-loaded H178A toward both substrates ranged from approximately 50- to 580-fold reduction. The pH dependence of log K(m), log k(cat), and log(k(cat)/K(m)) of both WT and H178A EcMetAP-I were also obtained and are identical, within error, for H178A and WT EcMetAP-I. Therefore, H178A is catalytically important but is not required for catalysis. Assignment of one of the observed pK(a) values at 8.1 for WT EcMetAP-I was obtained from plots of molar absorptivity at lambda(max(640)) vs pH for both WT and H178A EcMetAP-I. Apparent pK(a) values of 8.1 and 7.6 were obtained for WT and H178A EcMetAP-I, respectively, and were assigned to the deprotonation of a metal-bound water molecule. The data reported herein provide support for the key elements of the previously proposed mechanism and suggest that a similar mechanism can apply to the enzyme with a single metal in the active site.

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