Steady-state kinetics of Thr35 and Thr39 mutants in human adenylate kinase by site-directed mutagenesis.

Abstract

Adenylate kinase (AK; EC 2.7.4.3, hAK1) catalyzes the reaction: MgATP(2-)+ AMP2- reversible MgADP-(+) ADP3-. To elucidate the catalytic and structural roles of threonine residues in human AK, Thr35 and Thr39 mutants were analyzed by steady-state kinetics. The K(m) values of T35P and T35Y were not changed for MgATP2- and AMP2-, and the kcat values were decreased by 1/39 compared to those of wild-type AK. Thr35 was suggested to be essential for catalysis. The K(m) values of T39S, T39V and T39P were increased 5.6- to 59.0-fold for AMP2-; however, the kcat values were not reduced. Although the K(m) values of T39F and T39L were unchanged, the kcat values were reduced by more than 1/57. Thr39 appears to play an important role in the binding of AMP2- and to be essential for catalysis. As noted above, a hydroxyl group of the Thr residue in human AK appears to be important.