Kinetic and Spectroscopic Characterization of the H178A Methionyl Aminopeptidase from Escherichia coli
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- 作者：Alicja J. Copik ; Sabina I. Swierczek ; W. Todd Lowther ; Ventris M. D'souza ; Brian W. Matthews ; and Richard C. Holz
- 刊名：Biochemistry
- 出版年：2003
- 出版时间：May 27, 2003
- 年：2003
- 卷：42
- 期：20
- 页码：6283 - 6292
- 全文大小：182K
- 年卷期：v.42,no.20(May 27, 2003)
- ISSN：1520-4995
文摘
To gain insight into the role of the strictly conserved histidine residue, H178, in the reactionmechanism of the methionyl aminopeptidase from Escherichia coli (EcMetAP-I), the H178A mutant enzymewas prepared. Metal-reconstituted H178A binds only one equivalent of Co(II) or Fe(II) tightly with affinitiesthat 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 divalentmetal ion is pentacoordinate, identical to the WT enzyme. These data indicate that the metal binding sitehas not been affected by altering H178. The effect of altering H178 on activity is, in general, due to adecrease in kcat. The kcat value for Co(II)-loaded H178A decreased 70-fold toward MGMM and 290-foldtoward MP-p-NA compared to the WT enzyme, while kcat decreased 50-fold toward MGMM for theFe(II)-loaded H178A enzyme and 140-fold toward MP-p-NA. The Km values for MGMM remainedunaffected, while those for MP-p-NA increased approximately 2-fold for Co(II)- and Fe(II)-loaded H178A.The kcat/Km values for both Co(II)- and Fe(II)-loaded H178A toward both substrates ranged from ~50- to580-fold reduction. The pH dependence of log Km, log kcat, and log(kcat/Km) of both WT and H178AEcMetAP-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 pKavalues at 8.1 for WT EcMetAP-I was obtained from plots of molar absorptivity at 
max(640) vs pH for bothWT and H178A EcMetAP-I. Apparent pKa values of 8.1 and 7.6 were obtained for WT and H178AEcMetAP-I, respectively, and were assigned to the deprotonation of a metal-bound water molecule. Thedata reported herein provide support for the key elements of the previously proposed mechanism andsuggest that a similar mechanism can apply to the enzyme with a single metal in the active site.
max(640) vs pH for bothWT and H178A EcMetAP-I. Apparent pKa values of 8.1 and 7.6 were obtained for WT and H178AEcMetAP-I, respectively, and were assigned to the deprotonation of a metal-bound water molecule. Thedata reported herein provide support for the key elements of the previously proposed mechanism andsuggest that a similar mechanism can apply to the enzyme with a single metal in the active site.