Chemical mechanism of 6-phosphogluconate dehydrogenase from Candida utilis from pH studies.
The pH dependence of kinetic parameters and dissociation constants for competitive inhibitors was determined in order to obtain information on the chemical mechanism for the 6-phosphogluconate dehydrogenase reaction from Candida utilis. A mechanism is proposed in which an active site general base accepts the proton from the 3-hydroxyl concomitant with hydride transfer at C-3; the resulting 3-keto intermediate is decarboxylated to give the enol of ribulose 5-phosphate, followed by tautomerization of the enol to the keto product with the assist of a second enzyme residue acting as a general acid. There is also a requirement for an ionized phosphate of 6-phosphogluconate and ribulose 5-phosphate for optimum binding. The maximum velocity is pH dependent, decreasing at high and low pH giving pK values of 6 and 10, while the V/K for 6-phosphogluconate decreases at low pH with a slope of 2 yielding pK values of 6.4 and 7.6, respectively, and at high pH with a slope of -1 yielding a pK of 8.2. The 6-sulfogluconate pKi profile decreases at low and high pH giving pK values of 7.1 and 8.5, respectively. The 5-phosphoarabonate and 5-phosphoarabonate pKi profiles show similar behavior giving pK values of 6.5 and 8.8, respectively, for the former and 6.8 and 8.8, respectively, for the latter. The V/K for NADP also decreases at low and high pH giving pKs of 7.5 and 8.1, while the ATP-Ribose pKi profile decreases at low and high pH giving pKs of 7.2 and 8.0.(ABSTRACT TRUNCATED AT 250 WORDS)
Berdis
A J
AJ
Department of Biochemistry, Texas College of Osteopathic Medicine, Fort Worth 76107.
Cook
P F
PF
D002175:Candida / Q000201:enzymology*; D005942:Gluconates / Q000378:metabolism / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D010734:Phosphogluconate Dehydrogenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D005942:Gluconates; D010734:Phosphogluconate Dehydrogenase; C008884:6-phosphogluconic acid