Multiple catalytic activities of Escherichia coli lysyl-tRNA synthetase (LysU) are dissected by site-directed mutagenesis.

The heat-inducible lysyl-tRNA synthetase from Escherichia coli (LysU; EC6/1/1/6.html) converts ATP to diadenosine tri- and tetraphosphates (Ap(3)A/Ap(4)A) in the presence of L-lysine/Mg(2+)/Zn(2+). To understand LysU in more detail, 26 mutants were prepared: six of E264, four of R269 and sixteen mutants by alanine-scanning of the inner shell/motif 2 loop. In the presence of glycerol and absence of exogenously added Zn(2+)/L-lysine, we unexpectedly found that E264K catalysed the production of glycerol-3-phosphate, powered by ATP turnover to ADP. E264Q and E264N are also capable of this activity, but all three show little formation of Ap(4)A/Ap(3)A under normal conditions (additional Zn(2+)/L-lysine/Mg(2+)). By contrast, wild-type LysU has a weaker glycerol kinase-like capability in the absence of Zn(2+) and is dominated by Ap(4)A/Ap(3)A synthesis in its presence. Kinetic and isothermal titration calorimetry results suggest that E264 is a crucial residue for Zn(2+) promotion of Ap(4)A/Ap(3)A synthesis. This is consistent with the hypothesis that E264 provides an anchor point for a Zn(2+) ion complexed to the active site, with simultaneous coordination to the enzyme bound lysyl-adenylate intermediate and secondary substrate ATP/ADP. The glycerol kinase-like activity is uncovered on disruption of this specific coordination.