Transition state stabilization by six arginines clustered in the active site of creatine kinase.
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.
Jourden
Michael J
MJ
Department of Chemistry, College of Wooster, Wooster, OH 44691, USA.
Geiss
Paul R
PR
Thomenius
Michael J
MJ
Horst
Lindsay A
LA
Barty
Melissa M
MM
Brym
Melissa J
MJ
Mulligan
Guy B
GB
Almeida
Ryan M
RM
Kersteen
Betsy A
BA
Myers
Nichole R
NR
Snider
Mark J
MJ
Borders
Charles L
CL
Edmiston
Paul L
PL
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
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