Title

Chemical Mechanism of 6-phosphogluconate Dehydrogenase From Candida Utilis From pH Studies

Document Type

Article

Publication Date

3-1-1993

Publication Title

Biochemistry

Abstract

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-phosphoribonate 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. Studies of 6-phosphogluconate and ribulose 5-phosphate analogs provide information on the regiochemistry of the sugar-phosphate substrate for 6-phosphogluconate dehydrogenase. Basically, S stereochemistry at C-4 is required for optimum binding while R stereochemistry at C-2 is greatly preferred for substrate activity.

Comments

This work was supported by grants to P.F.C. from NIH (GM 36799) and the Robert A. Welch Foundation (B-1031).

DOI

10.1021/bi00059a022

Volume

32

Issue

8