Use of multiple isotope effects to determine enzyme mechanisms and intrinsic isotope effects. Malic enzyme and glucose 6-phosphate dehydrogenase

Abstract

By determining the 13C isotope effect on V/K with both a deuterated and an unlabeled substrate, and the deuterium isotope effect on V/K, it is possible to tell whether the 13C-sensitive and deuterium-sensitive steps are the same or not and, if they are different, to determine which comes first in the mechanism. If the two isotope-sensitive steps are the same, (1) deuteration increases the size of the observed 13C isotope effect, (2) narrow limits can be calculated for the intrinsic deuterium and 13C isotope effects, and (3) wider limits can be placed on the size of the commitments in the system. If an accurate determination of the tritium isotope effect on V/K is available, or if an a-secondary deuterium isotope effect which originates from the same step as the primary deuterium one and the corresponding 13C isotope effect with a-secondary deuterated substrate can be measured, an exact solution for the intrinsic isotope effects and the commitments is possible. With glucose-6-phosphate dehydrogenase, 13K, for label at C-1 of glucose is 0.9920, and 13C isotope effects are 1.0165, 1.03 16, and 1.01 76 with glucose 6-phosphate, glucose-I -d 6-phosphate, and glucose 6-phosphate (with TPN-4-4, respectively. The primary deuterium isotope effect on V/K of %e multistep nature of enzyme-catalyzed reactions usually decreases the magnitudes of observed isotope effects from the intrinsic isotope effects on the bond-breaking steps to somewhat lower values. Though this less than full expression can be a hindrance when trying to correlate isotope effects with transition-state structure, it can be quite useful in determining the relative rates of various steps and the sequence of these steps in a reaction mechanism. Often an isotopic substitution can be made which will affect the rate of only one particular step in a mechanism (typically via a primary deuterium isotope effect). By thus selectively changing the rate of this one step in the reaction mechanism and observing another isotope effect (either a deuterium or a heavy atom isotope effect) which is also expressed on a particular step of a mechanism, one can tell which isotope-sensitive step comes first in the mechanism or whether both isotope effects are on the same step. The concerted vs. stepwise controversy long associated with various enzymatic mechanisms can thus be unambiguously settled in many cases by application of the techniques which we will describe. We will show that the malic enzyme catalyzed oxidative decarboxylation of malate by triphosphopyridine nucleotide (TPN)' is a stepwise reaction with hydride transfer 2.97 and the a-secondary deuterium isotope effect of 1.00 allow calculation of intrinsic isotope effects and commitments in this system: Dk = 5.27, 13k = 1.0408, a-Dk = 1.054, cf = 0.75, and c, = 0.49. When deuterium-and 13C-sensitive steps are different, deuteration decreases the size of the observed I3C isotope effect. The data fit the equation [l3(V/K)H -I]/ [I3( V/K)D -11 = D(V/K)/DK, when the deuterium-sensitive step comes first but fit the equation...

pH dependence of the kinetic parameters for oxalacetate decarboxylation and pyruvate reduction reactions catalyzed by malic enzyme

“…1.1.1.38 (oxalacetate decarboxylating). Data presented here are consistent with oxalacetate as an intermediate in the reaction, as has already been shown for the NADP enzyme from chicken liver (Hermes et al, 1982). A proposed reaction mechanism for oxalacetate decarboxylation for the NAD and NADP enzyme is shown in Fig.…”

Molecular and Enzymatic Characterizations of Novel Bifunctional 3β-Hydroxysteroid Dehydrogenases/C-4 Decarboxylases from Arabidopsis thaliana

“…Extensive studies using multiple isotope effects have been carried out with a number of enzymes catalyzing oxidative decarboxylations with NADP ϩ as cosubstrate, exemplified by the malic enzyme (51)(52)(53) or the 6-phosphogluconate dehydrogenase (54). In these cases, a stepwise mechanism was shown, and a small isotope effect for the hydride transfer step was measured, comparable with that found with the 3␤HSD/Ds studied here.…”

Importance in Catalysis of the 6-Phosphate-binding Site of 6-Phosphogluconate in Sheep Liver 6-Phosphogluconate Dehydrogenase

“…13 C-Kinetic Isotope Effects-These effects were measured using the natural abundance of 13 C at C-1 of 6PG (12). Both high conversion (100% reaction, which represents 12 C/ 13 C in substrate) and low conversion samples were used to measure the 12 C/ 13 C isotope ratios in the CO 2 produced from the reaction of 3-h-6PG or 3-d-6PG.…”

“…Both high conversion (100% reaction, which represents 12 C/ 13 C in substrate) and low conversion samples were used to measure the 12 C/ 13 C isotope ratios in the CO 2 produced from the reaction of 3-h-6PG or 3-d-6PG. From these ratios, the 13 C-kinetic isotope effect was calculated (13).…”

“…Nomenclature-Isotope effects are expressed using the nomenclature developed by Northrop (19) and Cook and Cleland (20). Deuterium, 13 C, and solvent deuterium kinetic isotope effects are written with a leading superscript D, 13, or D 2 O, e.g. a primary deuterium isotope effect on V/K is written D (V/ K).…”