title: 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.
abstract: The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAH 7-P) synthase (Phe) is inactivated by diethyl pyrocarbonate (DEPC). The inactivation is first order with respect to enzyme and DEPC concentrations with a pseudo-second order rate constant of inactivation by DEPC of 4.9 +/- 0.8 m(-1) s(-1) at pH 6.8 and 4 degrees C. The dependence of inactivation on pH and the spectral features of enzyme modified at specific pH values imply that both histidine 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 against DEPC inactivation by phosphoenolpyruvate, whereas d-erythrose 4-phosphate 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 mutants retain some level of activity, whereas the H268G and H172G mutants are virtually inactive. A comparison of the circular dichroism spectra of wild-type enzyme and the various mutants demonstrates that H-172 may play a structural role. Comparison of the UV spectra of the H268G and wild-type enzymes saturated with Cu(2+) 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 studies. 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 of C-61 to glycine.
authors.affiliation: Department of Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor, Michigan 48109-1065, USA.
authors.forename: D L
authors.initials: DL
authors.lastname: Howe
authors.forename: H S
authors.initials: HS
authors.lastname: Duewel
authors.forename: R W
authors.initials: RW
authors.lastname: Woodard
mesh_terms: D010723:3-Deoxy-7-Phosphoheptulonate Synthase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000446:Aldehyde-Lyases / Q000378:metabolism*; D001483:Base Sequence; D002942:Circular Dichroism; D017931:DNA Primers; D006639:Histidine / Q000378:metabolism*; D007700:Kinetics; D008670:Metals / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation
chemical_list: D017931:DNA Primers; D008670:Metals; D006639:Histidine; D010723:3-Deoxy-7-Phosphoheptulonate Synthase; C020011:2-dehydro-3-deoxyphosphooctonate aldolase; D000446:Aldehyde-Lyases