Essential histidine residues in coenzyme B12-dependent diol dehydrase: dye-sensitized photooxidation and ethoxycarbonylation.
The apoenzyme of diol dehydrase was inactivated by photoirradiation in the presence of rose bengal or methylene blue, following pseudo-first-order kinetics. The inactivation rates were markedly reduced under a helium atmosphere, suggesting that the inactivation is due to photooxidation of the enzyme under air. The half-maximal rate of methylene blue-sensitized photoinactivation was observed at pH around 7.5. Amino acid analyses indicated that one to two histidine residues decreased upon the dye-sensitized photoinactivation, whereas the numbers of tyrosine, methionine, and lysine did not change. Ethoxyformic anhydride, another histidine-modifying reagent, also inactivated diol dehydrase, with pseudo-first-order kinetics and a half-maximal rate at pH 7.7. It was shown spectrophotometrically that three histidine residues per enzyme molecule were modified by this reagent with loss of enzyme activity. Two tyrosine residues per enzyme molecule were also modified rapidly, irrespective of the activity. The photooxidation or ethoxycarbonylation of the enzyme did not result in dissociation of the enzyme into subunits, but deprived the enzyme of ability to bind cyanocobalamin. The percentage loss of cobalamin-binding ability agreed well with the extent of inactivation. The enzyme-bound hydroxocobalamin showed only partial protecting effect against photoinactivation and resulting loss of the cobalamin-binding ability. These results provide evidence that diol dehydrase possesses essential histidine residues which are required for the coenzyme binding.
Kuno
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S
Department of Industrial Chemistry, Faculty of Engineering, Kyoto University, Japan.
Fukui
S
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Toraya
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T
D000596:Amino Acids / Q000032:analysis; D001051:Apoenzymes / Q000037:antagonists & inhibitors; D003038:Cobamides / Q000494:pharmacology*; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology*; D005561:Formates / Q000494:pharmacology*; D006639:Histidine; D006836:Hydro-Lyases / Q000037:antagonists & inhibitors*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000201:enzymology; D008027:Light; D046911:Macromolecular Substances; D008751:Methylene Blue / Q000494:pharmacology; D010084:Oxidation-Reduction; D010777:Photochemistry; D011408:Propanediol Dehydratase / Q000037:antagonists & inhibitors* / Q000378:metabolism / Q000528:radiation effects; D011485:Protein Binding; D012395:Rose Bengal / Q000494:pharmacology; D013053:Spectrophotometry
D000596:Amino Acids; D001051:Apoenzymes; D003038:Cobamides; D005561:Formates; D046911:Macromolecular Substances; D012395:Rose Bengal; D006639:Histidine; D006836:Hydro-Lyases; D011408:Propanediol Dehydratase; C000913:cobamamide; D004047:Diethyl Pyrocarbonate; D008751:Methylene Blue