The structural and functional roles of threonine-23 in the chicken muscle adenylate kinase (AK) were investigated by site-directed mutagenesis coupled with proton nuclear magnetic resonance (NMR) and phosphorus stereochemistry. The residue is potentially important because it is conserved among all types of AK and is part of the consensus P-loop sequence, 15GXPGXGKGT23. A mutant enzyme T23A (replacing threonine-23 with alanine) was constructed. Analyses of conformational stability and proton NMR indicate that the side chain of this residue contributes little 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 the mutant enzyme T23A showed no change in kcat and only 5-7-fold increases in Km and dissociation constants. Such minor changes in kinetic data are insufficient to suggest a functional role of Thr-23. However, two-dimensional NMR analyses of WT.MgAP5A and T23A.MgAP5A complexes indicated that the side chain of Thr-23 is in proximity to the adenine ring of the ATP moiety in the WT.MgAP5A complex in solution. In addition, T23A showed a significant perturbation in the stereospecificity toward the diastereomers of (Rp)- and (Sp)-adenosine 5'-(1-thiotriphosphate) (ATP alpha S), with the Rp/Sp ratio increased from < 0.02 in wild-type to 0.37 in T23A. Detailed 31P NMR analysis indicated that the stereospecificity at the AMP site was not perturbed. These results suggest that the side chain of Thr-23 is involved in catalysis, most likely via a hydrogen bonding interaction Thr-OH...O-P alpha(ATP).(ABSTRACT TRUNCATED AT 250 WORDS)