Identification of Essential Histidine Residues in 3-Deoxy-d-manno-octulosonic Acid 8-Phosphate Synthase:  Analysis by Chemical Modification with Diethyl Pyrocarbonate and Site-Directed Mutagenesis

• Published in: Biochemistry (Easton) 1999-10, Vol.38 (43), p.14320-14329
• Main Authors: Sheflyan, Galina Ya, Duewel, Henry S, Chen, Grace, Woodard, Ronald W
• Format: Article
• Language: English
• Subjects: Aldehyde-Lyases - antagonists & inhibitors; Aldehyde-Lyases - chemistry; Aldehyde-Lyases - genetics; Aldehyde-Lyases - metabolism; Amino Acid Sequence; Binding Sites; Catalysis; Diethyl Pyrocarbonate - pharmacology; Enzyme Activation - drug effects; Escherichia coli - enzymology; Escherichia coli - genetics; Histidine - chemistry; Histidine - genetics; Histidine - metabolism; Hydrogen-Ion Concentration; imidazole; Kinetics; Mutagenesis, Site-Directed; Substrate Specificity
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi9829884

The enzyme 3-deoxy-d-manno-octulosonic acid 8-phosphate (KDO 8-P) synthase from Escherichia coli that catalyzes the aldol-type condensation of d-arabinose 5-phosphate (A 5-P) and phosphoenolpyruvate (PEP) to give KDO 8-P and inorganic phosphate (Pi) is inactivated by diethyl pyrocarbonate (DEPC). The inactivation is first-order in enzyme and DEPC. A second-order rate constant of 340 M-1 min-1 is obtained at pH 7.6 and 4 °C. The rate of inactivation is dependent on pH and the pH-inactivation rate data imply the involvement of an amino acid residue with a pK a value of 7.3. KDO 8-P synthase activity is not restored to the DEPC-inactivated enzyme following treatment with hydroxylamine. Complete loss of KDO 8-P synthase activity correlates with the ethoxyformylation of three histidine residues by DEPC. KDO 8-P synthase is protected against DEPC inactivation by PEP and partially protected against inactivation by A 5-P. To provide further evidence for the involvement or role of the histidine residues in the aldol-type condensation catalyzed by KDO 8-P synthase, all six histidines were individually mutated to either glycine or alanine. The kinetic constants for the three mutants H40A, H67G, and H246G were unaffected as compared to the wild type enzyme. In contrast, H241G demonstrates a >10-fold increase in K M for both PEP and A 5-P and a 4-fold reduction in k cat, while H97G demonstrates an increase in K M for only A 5-P and a 2-fold reduction in k cat. The activity of the H202G mutant was too low to be measured accurately but the data obtained indicated an approximate 400-fold reduction in k cat. Circular dichroism measurements of the wild-type and mutant enzymes indicate modest structural changes in only the fully active H67G and H246G mutants. The H241G mutant is protected against DEPC inactivation by PEP and A 5-P to the same extent as the wild-type enzyme, suggesting that the functionally important H241 may not be located in the vicinity of the substrate binding sites. The H97G mutant is protected by PEP against DEPC inactivation to the same degree as the wild-type enzyme but is no longer protected by A 5-P. In the case of the H202G mutant, both A 5-P and PEP protect the mutant against DEPC inactivation but to different extents from those observed for the wild-type enzyme. The catalytic activity of the H97G mutant is partially restored (20% → 60% of wild-type activity) in the presence of imidazole, while a minor amount of activity is restored to the H202G m