Pyridoxal kinase catalyzes the phosphorylation of pyridoxal (PL) to pyridoxal 5'-phosphate (PLP). A D235A variant shows 7-fold and 15-fold decreases in substrate affinity and activity, respectively. A D235N variant shows approximately 2-fold decrease in both PL affinity and activity. The crystal structure of D235A (2.5 A) shows bound ATP, PL and PLP, while D235N (2.3 A) shows bound ATP and sulfate. These results document the role of Asp235 in PL kinase activity. The observation that the active site of PL kinase can accommodate both ATP and PLP suggests that formation of a ternary Enz.PLP.ATP complex could occur in the wild-type enzyme, consistent with severe MgATP substrate inhibition of PL kinase in the presence of PLP.