STRUCTURALLY AND CATALYTICALLY IMPORTANT RESIDUES IN THE PHOSPHATE BINDING LOOP OF ADENYLATE KINASE OF ESCHERICHIA-COLI

被引:102
作者
REINSTEIN, J
SCHLICHTING, I
WITTINGHOFER, A
机构
[1] Abteilung Biophysik, Max-Planck-Institut für medizinische Forschung, 6900 Heidelberg
关键词
D O I
10.1021/bi00484a014
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Amino acids in the phosphate binding loop of adenylate kinase of Escherichia coli were mutated by site-directed mutagenesis. The mutant proteins with a Pro-9 → Gly (P9G) and with a Lys-13 → Gin (K13Q) exchange were overexpressed and purified. They were characterized by steady-state kinetics, fluorescence binding, and structural studies, together with the phosphate binding loop mutants P9L and G10V prepared earlier [Reinstein, J., Brune, M., & Wittinghofer, A. (1988) Biochemistry 27, 4712-4720]. The results obtained show that all these mutations change the structure of the protein as evidenced by NMR spectroscopy and temperature-stability studies. All the mutant proteins have increased dissociation constants for substrates and inhibitors, but their catalytic activity, except for K13Q, is not reduced. The results obtained with K13Q suggest that this lysine residue, which is conserved in all guanine and many adenine nucleotide proteins, might have an important role in catalysis. © 1990, American Chemical Society. All rights reserved.
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页码:7451 / 7459
页数:9
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