title: Transition state stabilization by six arginines clustered in the active site of creatine kinase.
abstract: Six fully conserved arginine residues (R129, R131, R235, R291, R319, and R340) closely grouped in the nucleotide binding site of rabbit muscle creatine kinase (rmCK) were mutated; four to alanine and all six to lysine. Kinetic analyses in the direction of phosphocreatine formation showed that all four alanine mutants led to substantial losses of activity with three (R129A, R131A, and R235A) having no detectable activity. All six lysine mutants retained variable degrees of reduced enzymatic activity. Static quenching of intrinsic tryptophan fluorescence was used to measure the binding constants for MgADP and MgATP. Nucleotide binding was at most only modestly affected by mutation of the arginine residues. Thus, the cluster of arginines seem to be primarily responsible for transition state stabilization which is further supported by the observation that none of the inactive mutants demonstrated the ability to form a transition analogue complex of MgADP.nitrate.creatine as determined by fluorescence quenching assays. As a whole, the results suggest that the most important role these residues play is to properly align the substrates for stabilization of the phosphoryl transfer reaction.
authors: Department of Chemistry, College of Wooster, Wooster, OH 44691, USA.
authors: Michael J
authors: MJ
authors: Jourden
authors: Paul R
authors: PR
authors: Geiss
authors: Michael J
authors: MJ
authors: Thomenius
authors: Lindsay A
authors: LA
authors: Horst
authors: Melissa M
authors: MM
authors: Barty
authors: Melissa J
authors: MJ
authors: Brym
authors: Guy B
authors: GB
authors: Mulligan
authors: Ryan M
authors: RM
authors: Almeida
authors: Betsy A
authors: BA
authors: Kersteen
authors: Nichole R
authors: NR
authors: Myers
authors: Mark J
authors: MJ
authors: Snider
authors: Charles L
authors: CL
authors: Borders
authors: Paul L
authors: PL
authors: Edmiston
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000409:Alanine / Q000737:chemistry / Q000235:genetics; D000818:Animals; D001120:Arginine / Q000737:chemistry / Q000235:genetics*; D020134:Catalytic Domain / Q000235:genetics*; D003401:Creatine / Q000737:chemistry; D003402:Creatine Kinase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D052283:Creatine Kinase, MM Form; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008239:Lysine / Q000737:chemistry / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010725:Phosphocreatine / Q000737:chemistry; D011485:Protein Binding; D011817:Rabbits; D013050:Spectrometry, Fluorescence
chemical_list: D007527:Isoenzymes; D010725:Phosphocreatine; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D001120:Arginine; D003402:Creatine Kinase; D052283:Creatine Kinase, MM Form; D008239:Lysine; D003401:Creatine; D000409:Alanine