Histidine 268 in 3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthase plays the same role as histidine 202 in 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase

The enzyme 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH 7-P) synthase ( Phe) is inactivated by diethyl pyrocarbonate (DEPC). The inactivation is fi rst order with respect to enzyme and DEPC concentrations with a pseudo-seco nd order rate constant of inactivation by DEPC of 4.9 +/- 0.8 M-1 s(-1) at pH 6.8 and 4 degreesC. The dependence of inactivation on pH and the spectra l features of enzyme modified at specific pH values imply that both histidi ne and cysteine residues are modified, which is confirmed by site-directed mutagenesis. Analysis of the chemical modification data indicates that one histidine is essential for activity. DAH 7-P synthase (Phe) is protected ag ainst DEPC inactivation by phosphoenolpyruvate, whereas D-erythrose 4-phosp hate offers only minimal protection. The conserved residues H-172, H-207, H -268, and H-304 were individually mutated to glycine. The H304G and H207G m utants retain some level of activity, whereas the H268G and H172G mutants a re virtually inactive. A comparison of the circular dichroism spectra of wi ld-type enzyme and the various mutants demonstrates that H-172 may play a s tructural role. Comparison of the UV spectra of the H268G and wildtype enzy mes saturated with Cu2+ indicates that the metal-binding site of the H268G mutant resembles that of the wild-type enzyme. The residue H-268 may play a catalytic role based on the site-directed mutagenesis and spectroscopic st udies. Cysteine 61 appears to influence the pK(a) of H-268 in the wild-type enzyme. The pK(a), of H-268 increases from 6.0 to 7.0 following mutation o f C-61 to glycine.