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Asparagine 285 plays a key role in transition state stabilization in rabbit muscle creatine kinase

Department of Chemistry, The College of Wooster, Wooster, Ohio 44691, USA

Reprint requests to: Charles L. Borders, Jr., Department of Chemistry, College of Wooster, Wooster, OH 44691, USA; e-mail: borders{at}wooster.edu; fax: (330) 263-2386.

To explore the possibility that asparagine 285 plays a key role in transition state stabilization in phosphagen kinase catalysis, the N285Q, N285D, and N285A site-directed mutants of recombinant rabbit muscle creatine kinase (rmCK) were prepared and characterized. Kinetic analysis of phosphocreatine formation showed that the catalytic efficiency of each N285 mutant was reduced by approximately four orders of magnitude, with the major cause of activity loss being a reduction in k_cat in comparison to the recombinant native CK. The data for N285Q still fit a random-order, rapid-equilibrium mechanism, with either MgATP or creatine binding first with affinities very nearly equal to those for native CK. However, the affinity for the binding of the second substrate is reduced approximately 10-fold, suggesting that addition of a single methylene group at position 285 disrupts the symphony of substrate binding. The data for the N285A mutant only fit an ordered binding mechanism, with MgATP binding first. Isosteric replacement to form the N285D mutant has almost no effect on the K_M values for either creatine or MgATP, thus the decrease in activity is due almost entirely to a 5000-fold reduction in k_cat. Using the quenching of the intrinsic CK tryptophan fluorescence by added MgADP (Borders et al. 2002), it was found that, unlike native CK, none of the mutants have the ability to form a quaternary TSAC. We use these data to propose that asparagine 285 indeed plays a key role in transition state stabilization in the reaction catalyzed by creatine kinase and other phosphagen kinases.

Keywords: Creatine kinase; asparagine 285; mutagenesis; transition state stabilization

Abbreviations: CK, creatine kinase • AK, arginine kinase • TSAC, transition-state analog complex

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