A novel dehydrogenase reaction mechanism for hexose-6-phosphate dehydrogenase isolated from the teleost Fundulus heteroclitus.
Hexose-6-phosphate dehydrogenase (refers to hexose-6-phosphate dehydrogenase from any species in general) has been purified to apparent homogeneity from the teleost fish Fundulus heteroclitus. The enzyme was characterized for native (210 kDa) and subunit molecular mass (54 kDa), isoelectric point (6.65), amino acid composition, substrate specificity, and metal dependence. Glucose 6-phosphate, galactose 6-phosphate, 2-deoxyglucose 6-phosphate, glucose 6-sulfate, glucosamine 6-phosphate, and glucose were found to be substrates in the reaction with NADP+, but only glucose was a substrate when NAD+ was used as coenzyme. A unique reaction mechanism for the forward direction was found for this enzyme when glucose 6-phosphate and NADP+ were used as substrates; ordered with glucose 6-phosphate binding first. NAD+ was found to be a competitive inhibitor toward NADP+ and an uncompetitive inhibitor with regard to glucose 6-phosphate in this reaction; Vmax = 7.56 mumol/min/mg, Km(NADP+) = 1.62 microM, Km(glucose 6-phosphate) = 7.29 microM, Kia(glucose 6-phosphate) = 8.66 microM, and Ki(NAD+) = 0.49 microM. The use of alternative substrates confirmed this result. This type of reaction mechanism has not been previously reported for a dehydrogenase.
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McCollum-Pratt Institute, Johns Hopkins University, Baltimore, Maryland 21218.
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D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002237:Carbohydrate Dehydrogenases / Q000302:isolation & purification / Q000378:metabolism*; D005399:Fishes / Q000378:metabolism*; D019298:Glucose-6-Phosphate; D005958:Glucosephosphates / Q000378:metabolism; D007526:Isoelectric Point; D007700:Kinetics; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D013379:Substrate Specificity
D000596:Amino Acids; D005958:Glucosephosphates; D009243:NAD; D009249:NADP; D019298:Glucose-6-Phosphate; D002237:Carbohydrate Dehydrogenases; C020552:galactose-6-phosphate dehydrogenase