TAILIEUCHUNG - Báo cáo khoa học: Analyzing the catalytic role of Asp97 in the methionine aminopeptidase from Escherichia coli

An active site aspartate residue, Asp97, in the methionine aminopeptidase (MetAPs) from Escherichia coli(EcMetAP-I) was mutated to alanine, glu-tamate, and asparagine. Asp97 is the lone carboxylate residue bound to the crystallographically determined second metal-binding site in EcMetAP-I. | Analyzing the catalytic role of Asp97 in the methionine aminopeptidase from Escherichia coli Sanghamitra Mitra1 Kathleen M. Job1 Lu Meng1 Brian Bennett2 and Richard C. Holz1 3 1 Department of Chemistry and Biochemistry Utah State University Logan UT USA 2 Department of Biophysics NationalBiomedicalEPR Center MedicalCollege of Wisconsin Milwaukee WI USA 3 Department of Chemistry Loyola University-Chicago IL USA Keywords EPR kinetics mechanism methionine aminopeptidases mutants Correspondence R. C. Holz Department of Chemistry Loyola University-Chicago 1068 West Sheridan Road Chicago IL 60626 USA Fax 1 773 508 3086 Tel 1 773 508 3092 E-mail rholz1@ Received 9 September 2008 revised 13 October 2008 accepted 17 October 2008 doi An active site aspartate residue Asp97 in the methionine aminopeptidase MetAPs from Escherichia coli EcMetAP-I was mutated to alanine glutamate and asparagine. Asp97 is the lone carboxylate residue bound to the crystallographically determined second metal-binding site in EcMetAP-I. These mutant EcMetAP-I enzymes have been kinetically and spectroscopically characterized. Inductively coupled plasma-atomic emission spectroscopy analysis revealed that equivalents of cobalt were associated with each of the Asp97-mutated EcMetAP-Is. The effect on activity after altering Asp97 to alanine glutamate or asparagine is in general due to a 9000-fold decrease in kca towards Met-Gly-Met-Met as compared to the wild-type enzyme. The Co II d-d spectra for wild-type D97E and D97A EcMetAP-I exhibited very little difference in form in each case between the monocobalt II and dicobalt II EcMetAP-I and only a doubling of intensity was observed upon addition of a second Co II ion. In contrast the electronic absorption spectra of Co_ D97N EcMetAP-I and CoCo D97N EcMetAP-I were distinct as were the EPR spectra. On the basis of the observed molar absorptivities the Co II ions binding to the D97E D97A and D97N EcMetAP-I active

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