MECHANISM OF ADENYLATE KINASE - WHAT CAN BE LEARNED FROM A MUTANT ENZYME WITH MINOR PERTURBATION IN KINETIC-PARAMETERS

The structural and functional roles of threonine-23 in the chicken mus cle adenylate kinase (AK) were investigated by site-directed mutagenes is coupled with proton nuclear magnetic resonance (NMR) and phosphorus stereochemistry. The residue is potentially important because it is c onserved among all types of AK and is part of the consensus P-loop seq uence, 15GXPGXGKGT23. A mutant enzyme T23A (replacing threonine-23 wit h alanine) was constructed. Analyses of conformational stability and p roton NMR indicate that the side chain of this residue contributes lit tle to the structure of AK, which suggests that the side chain of Thr- 23 does not play a structural role. The steady-state kinetic data of t he mutant enzyme T23A showed no change in k(cat) and only 5-7-fold inc reases in K(m) and dissociation constants. Such minor changes in kinet ic data are insufficient to suggest a functional role of Thr-23. Howev er, two-dimensional NMR analyses of WT.MgAP5A and T23A.MgAP5A complexe s indicated that the side chain of Thr-23 is in proximity to the adeni ne ring of the ATP moiety in the WT-MgAP5A complex in solution. In add ition, T23A showed a significant perturbation in the stereospecificty toward the diastereomers of (R(p))- and (S(p))-adenosine 5'-(1-thiotri phosphate) (ATPalphaS), with the R(p)/S(p) ratio increased from <0.02 in wild-type to 0.37 in T23A. Detailed P-31 NMR analysis indicated tha t the stereospecificity at the AMP site was not perturbed. These resul ts suggest that the side chain of Thr-23 is involved in catalysis, mos t likely via a hydrogen bonding interaction Thr-OH...O-P(alpha)(ATP). Thus, even though it is not obvious from the kinetic data of the mutan t T23A, the side chain of Thr-23 does interact directly with ATP durin g catalysis. The significance of these results is discussed in relatio n to the structure-function relationship of adenylate kinase in partic ular and the use of site-directed mutagenesis in the study of enzyme m echanism in general.