Use of intermediate partitioning to calculate intrinsic isotope effects for the reaction catalyzed by malic enzyme.

For those enzymes that proceed via a stepwise reaction mechanism with a discrete chemical intermediate and where deuterium and 13C isotope effects are on separate steps, a new method has been developed to solve for the intrinsic deuterium and 13C kinetic isotope effects that relies on directly observing the partitioning of the intermediate between the forward and reverse directions. This observed partitioning ratio, along with the values of the primary deuterium, tritium, and 13C kinetic isotope effects on V/K for the substrate with the label being followed, allows an exact solution for the intrinsic deuterium and 13C isotope effects, the forward commitment for the deuterium-sensitive step, and the partition ratio for the intermediate in the reaction. This method allows portions of the reaction coordinate diagram to be defined precisely and the relative energy levels of certain activation barriers to be assigned exactly. With chicken liver triphosphopyridine nucleotide (TPN) malic enzyme activated by Mg2+, the partitioning of oxalacetate to pyruvate vs. malate in the presence of TPNH, 0.47, plus previously determined isotope effects gives an intrinsic deuterium isotope effect of 5.7 on hydride transfer and a 13C isotope effect of 1.044 on decarboxylation. Reverse hydride transfer is 10 times faster than decarboxylation, and the forward commitment for hydride transfer is 3.3. The 13C isotope effect is not significantly different with reduced acetylpyridine adenine dinucleotide phosphate replacing TPNH (although the pyruvate/malate partitioning ratio for oxalactate is now 9.9), but replacement of Mg2+ by Mn2+ raises the value to 1.065 (partition ratio 0.99).