6-Phosphogluconate dehydrogenase mechanism: evidence for allosteric modulation by substrate

Abstract

The reductive carboxylation of ribulose-5-phosphate (Ru5P) by 6-phosphogluconate dehydrogenase (6PGDH) from Candida utilis was investigated using kinetic isotope effects. The intrinsic isotope effect for proton abstraction from Ru5P was found at 4.9 from deuterium isotope effects on V and V/K and from tritium isotope effects on V/K. The presence of 6-phosphogluconate (6PG) in the assay mixture changes the magnitude of the observed isotope effects. In the absence of 6PG (D)(V/K) and (D)(V) are 1.68 and 2.46, respectively, whereas the presence of 6PG increases (D)(V/K) to 2.84 and decreases (D)(V) to 1.38. A similar increase of (T)(V/K) is observed as 6PG builds up in the reaction mixture. These data indicate that in the absence of 6PG, a slow step, which precedes the chemical process, is rate-limiting for the reaction, whereas in the presence of 6PG, the rate-limiting step follows the isotope-sensitive step. Kinetic analysis of reductive carboxylation shows that 6PG at low concentrations decreases the K(m) of Ru5P, whereas at higher concentrations, the usual competitive pattern is observed. These data indicate that full activity of 6PGDH is achieved when one subunit carries out the catalysis and the other subunit carries an unreacted 6PG. Thus, 6PG is like an allosteric activator of 6PGDH.

SCHEME 1.

6PGDH-catalyzed reaction and the two main amino acid residues involved.

SCHEME 2.

The overall kinetic mechanism of 6PGDH.

FIGURE 1.

Dependence of the calculated tritium isotope effect on the extent of the reaction. ^T(V/K) was calculated from the change in Ru5P specific activity at different conversion grades from Ru5P to 6PG (Equation 3).

FIGURE 2.

Effects of 6PG on the reductive carboxylation of Ru5P. Effects of 6PG on K_app, where K_app = K_Ru5P(1 + [6PG]/K_i6PG). Squares, experimental points; line: interpolated curve assuming that K_i6PG = 11 μm, K_Ru5P = 287 μm in the absence of 6PG and 117 μm in the presence of 6PG.

FIGURE 3.

Cooperative versus Michaelian behavior of 6PGDH. Squares, cooperative behavior assuming K_m 70 μm and V_max 1.0 for the dimeric enzyme with one 6PG bound, and K_m 44 μm and V_max 1.0 for the dimeric enzyme with two 6PG bound; line: nonlinear fit according to Michaelis-Menten kinetics giving V_max 1.01 and K_m 38 μm.