Mechanism of adenylate kinase. The conserved aspartates 140 and 141 are important for transition state stabilization instead of substrate-induced conformational changes.
The goal of this work is to evaluate the quantitative contribution of Asp-140 and Asp-141 to conformational changes and/or substrate stabilization in the catalysis by chicken muscle adenylate kinase (AK), by use of kinetic and structural analysis of the single alanine mutants D140A and D141A as well as the salt bridge double mutants R138M,D140A and R132M,D141A. The single mutants D140A and D141A displayed small increases in Km (6-12-fold) and moderate decreases in kcat (17-78-fold). Kinetic analysis with the slowly reacting substrate system MgATP and dAMP suggested that the decrease in kcat is not caused by a decrease in the conformational step(s) relative to the chemical step. Most of the kinetic constants of the double mutants R138M,D140A and R132M,D141A are similar to those of the single mutants R138M and R132M, respectively. Detailed analysis by two-dimensional NMR indicated no appreciable changes in the conformations of the free enzyme or the complex with MgAP5A (where AP5A is P1,P5-bis(5'-adenosyl)pentaphosphate), for both single and double mutants. These results taken together suggest that Asp-140 and Asp-141 are unimportant for substrate-induced conformational changes in AK; their roles are mainly to assist Arg-138 and Arg-132 in stabilizing the transition state. The structural results also suggest that AK is a flexible enzyme.
Dahnke
T
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Department of Chemistry, Ohio State University, Columbus 43210.
Tsai
M D
MD
D000218:Adamantane / Q000031:analogs & derivatives / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000263:Adenylate Kinase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid; D001483:Base Sequence; D001665:Binding Sites; D002645:Chickens; D003001:Cloning, Molecular; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D019791:Guanidine; D006146:Guanidines; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013816:Thermodynamics
D003838:Deoxyadenine Nucleotides; D006146:Guanidines; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D001224:Aspartic Acid; C016358:2,4-diamino-5-adamantyl-6-methylpyrimidine; C012873:2'-deoxy-5'-adenosine monophosphate; D000255:Adenosine Triphosphate; D000263:Adenylate Kinase; D019791:Guanidine; D000218:Adamantane