STEADY-STATE KINETICS OF THR35 AND THR39 MUTANTS IN HUMAN ADENYLATE KINASE BY SITE-DIRECTED MUTAGENESIS

Adenylate kinase (AK; EC 2.7.4.3, hAK1) catalyzes the reaction: MgATP( 2-) + AMP(2-) reversible arrow MgADP(-) + ADP(3-). To elucidate the ca talytic and structural roles of threonine residues in human AK, Thr35 and Thr39 mutants were analyzed by steady-state kinetics. The K-m valu es of T35P and T35Y were not changed for MgATP(2-) and AMP(2-), and th e k(cat) 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 AMP(2-); however, the k(cat) values were not reduced. Although the K-m values o f T39F and T39L were unchanged, the k(cat) values were reduced by more than 1/57. Thr39 appears to play an important role in the binding of AMP(2-) and to be essential for catalysis. As noted above, a hydroxyl group of the Thr residue in human AK appears to be important.