title: Mechanism of adenylate kinase. Structural and functional roles of the conserved arginine-97 and arginine-132.
abstract: The structural and functional roles of two conserved active site residues, Arg-97 and Arg-132, in chicken muscle adenylate kinase (AK) were evaluated by site-directed mutagenesis in conjunction with one- and two-dimensional proton nuclear magnetic resonance (NMR), kinetics, and guanidine hydrochloride-induced denaturation. In addition, 31P NMR analysis was used to evaluate the contribution of Arg-97 to the phosphorus stereospecificity of AK. The results and conclusions are summarized as follows: (i) Kinetic analysis of R97M reveals 6- and 28-fold increases in the dissociation constant Ki and Michaelis constant K of AMP, respectively, and a moderate 30-fold decrease in kcat. The Ki and K values of MgATP are relatively unperturbed. The localized effect of AMP stabilization was independently confirmed by proton NMR titration, which showed a ca. 20-fold increase in the dissociation constant of AMP but not of MgATP. (ii) R132M affords a dramatic decrease in kcat by a factor of 8.0 x 10(3), with unchanged dissociation and Michaelis constants for either substrate. The lack of perturbation in the affinities toward substrates was confirmed by proton NMR titration. (iii) Although small chemical shift changes were observed for the free mutants and their complexes with substrates, further analyses by nuclear Overhauser enhanced spectroscopy with the bisubstrate analogue inhibitor, P1,P5-bis(5'-adenosyl)pentaphosphate (AP5A), indicated little perturbation in the global conformation. (iv) Contributions to conformational stability by Arg-97 and Arg-132 are negligible on the basis of the free energy of unfolding, delta GdH2O. (v) R97M was predicted and demonstrated to exhibit enhanced stereospecificity at the AMP site by at least 10-fold relative to WT in the conversion of adenosine 5'-monothiophosphate to adenosine 5'-(1-thiodiphosphate). This result for R97M was predicted on the basis of the orientation of Arg-97 relative to Arg-44 and AMP in the active site as observed in available crystal structures and the stereospecificity results of R44M [Jiang, R.-T., Dahnke, T., & Tsai, M.-D. (1991) J. Am. Chem. Soc. 113, 5485-5486]. (vi) The above structural and functional analyses led us to conclude that Arg-97 interacts with the phosphoryl group of AMP, beginning at the binary complex (1-2 kcal/mol), continuing through the transition state (3.5 kcal/mol), and that Arg-132 stabilizes the transition state by greater than 5 kcal/mol. (vii) The functional importance of Arg-97 appears to be similar to that of Arg-44 [Yan, H., Dahnke, T., Zhou, B., Nakazawa, A., & Tsai, M.-D. (1990) Biochemistry 29, 10956-10964].(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Chemistry, Ohio State University, Columbus 43210.
authors.forename: T
authors.initials: T
authors.lastname: Dahnke
authors.forename: Z
authors.initials: Z
authors.lastname: Shi
authors.forename: H
authors.initials: H
authors.lastname: Yan
authors.forename: R T
authors.initials: RT
authors.lastname: Jiang
authors.forename: M D
authors.initials: MD
authors.lastname: Tsai
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000263:Adenylate Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine; D001483:Base Sequence; D001665:Binding Sites; D002645:Chickens; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010758:Phosphorus; D011487:Protein Conformation
chemical_list: D009838:Oligodeoxyribonucleotides; D010758:Phosphorus; D000244:Adenosine Diphosphate; D001120:Arginine; D000263:Adenylate Kinase