Mannitol-1-phosphate dehydrogenase of Escherichia coli. Chemical properties and binding of substrates.
Mannitol-1-phosphate dehydrogenase was purified to homogeneity, and some chemical and physical properties were examined. The isoelectric point is 4.19. Amino acid analysis and polyacrylamide-gel electrophoresis in presence of SDS indicate a subunit Mr of about 22,000, whereas gel filtration and electrophoresis of the native enzyme indicate an Mr of 45,000. Thus the enzyme is a dimer. Amino acid analysis showed cysteine, tyrosine, histidine and tryptophan to be present in low quantities, one, three, four and four residues per subunit respectively. The zinc content is not significant to activity. The enzyme is inactivated (greater than 99%) by reaction of 5,5'-dithiobis-(2-nitrobenzoate) with the single thiol group; the inactivation rate depends hyperbolically on reagent concentration, indicating non-covalent binding of the reagent before covalent modification. The pH-dependence indicated a pKa greater than 10.5 for the thiol group. Coenzymes (NAD+ and NADH) at saturating concentrations protect completely against reaction with 5,5'-dithiobis-(2-nitrobenzoate), and substrates (mannitol 1-phosphate, fructose 6-phosphate) protect strongly but not completely. These results suggest that the thiol group is near the catalytic site, and indicate that substrates as well as coenzymes bind to free enzyme. Dissociation constants were determined from these protective effects: 0.6 +/- 0.1 microM for NADH, 0.2 +/- 0.03 mM for NAD+, 9 +/- 3 microM for mannitol 1-phosphate, 0.06 +/- 0.03 mM for fructose 6-phosphate. The binding order for reaction thus may be random for mannitol 1-phosphate oxidation, though ordered for fructose 6-phosphate reduction. Coenzyme and substrate binding in the E X NADH-mannitol 1-phosphate complex is weaker than in the binary complexes, though in the E X NADH+-fructose 6-phosphate complex binding is stronger.
D000596:Amino Acids / Q000032:analysis; D004228:Dithionitrobenzoic Acid / Q000037:antagonists & inhibitors; D004926:Escherichia coli / Q000201:enzymology*; D005636:Fructosephosphates / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008355:Mannitol Phosphates / Q000378:metabolism; D008970:Molecular Weight; D013401:Sugar Alcohol Dehydrogenases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D015032:Zinc / Q000032:analysis
D000596:Amino Acids; D005636:Fructosephosphates; D046911:Macromolecular Substances; D008355:Mannitol Phosphates; C030070:mannitol-1-phosphate; C032284:fructose-1-phosphate; D004228:Dithionitrobenzoic Acid; D013401:Sugar Alcohol Dehydrogenases; C021544:mannitol-1-phosphate dehydrogenase; D015032:Zinc