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.

Generation of an active monomer of rabbit muscle creatine kinase by site-directed mutagenesis: the effect of quaternary structure on catalysis and stability.

Cytosolic creatine kinase exists in native form as a dimer; however, the reasons for this quaternary structure are unclear, given that there is no evidence of active site communication and more primitive guanidino kinases are monomers. Three fully conserved residues found in one-half of the dimer interface of the rabbit muscle creatine kinase (rmCK) were selectively changed to alanine by site-directed mutagenesis. Four mutants were prepared, overexpressed, and purified: R147A, R151A, D209A, and R147A/R151A. Both the R147A and R147A/R151A were confirmed by size-exclusion chromatography and analytical ultracentrifugation to be monomers, whereas R151A was dimeric and D209A appeared to be an equilibrium mixture of dimers and monomers. Kinetic analysis showed that the monomeric mutants, R147A and R147A/R151A, showed substantial enzymatic activity. Substrate binding affinity by R147A/R151A was reduced approximately 10-fold, although k(cat) was 60% of the wild-type enzyme. Unlike the R147A/R151A, the kinetic data for the R147A mutant could not be fit to a random-order rapid-equilibrium mechanism characteristic of the wild-type, but could only be fit to an ordered mechanism with creatine binding first. Substrate binding affinities were also significantly lower for the R147A mutant, but k(cat) was 11% that of the native enzyme. Fluorescence measurements using 1-anilinonaphthalene-8-sufonate showed that increased amounts of hydrophobic surface area are exposed in all of the mutants, with the monomeric mutants having the greatest amounts of unfolding. Thermal inactivation profiles demonstrated that protein stability is significantly decreased in the monomeric mutants compared to wild-type. Denaturation experiments measuring lambda(max) of the intrinsic fluorescence as a function of guanidine hydrochloride concentration helped confirm the quaternary structures and indicated that the general unfolding pathway of all the mutants are similar to that of the wild-type. Collectively, the data show that dimerization is not a prerequisite for activity, but there is loss of structure and stability upon formation of a CK monomer.