Kinetic and Spectroscopic Characterization of the H178A Methionyl Aminopeptidase from Escherichia coli

Authors

Alicja J. Copik, Utah State University
Sabina I. Swierczek, Utah State UniversityFollow
W. Todd Lowther, University of Oregon
Ventris M. D'Souza, Utah State University
Brian W. Matthews, University of Oregon
Richard C. Holz, Marquette UniversityFollow

Document Type

Article

Publication Date

5-3-2003

Source Publication

Biochemistry

Source ISSN

0006-2960

Abstract

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 ∼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 λ_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.

Comments

Accepted version. Biochemistry, Vol. 42, No. 20 (May 3, 2003): 6283-6292. DOI. © 2003 American Chemical Society. Used with permission.

Richard C. Holz was affiliated with Utah State University at the time of publication.

Recommended Citation

Copik, Alicja J.; Swierczek, Sabina I.; Lowther, W. Todd; D'Souza, Ventris M.; Matthews, Brian W.; and Holz, Richard C., "Kinetic and Spectroscopic Characterization of the H178A Methionyl Aminopeptidase from Escherichia coli" (2003). Chemistry Faculty Research and Publications. 315.
https://epublications.marquette.edu/chem_fac/315