- Deuterium Isotope Effects for the Nonenzymatic and Glutamate Dehydrogenase Catalyzed Reduction of an α-Imino Acid by NADH
-
The mechanisms of the nonenzymatic and glutamate dehydrogenase catalyzed reduction of an α-imino acid, Δ1-pyrroline-2-carboxylic acid, by NAD(P)H have been studied by deuterium isotope effects.The partition isotope effects for the nonenzymatic reaction with 4-deuterated 1,4-dihydronicotinamides are about the same as the corresponding observed kinetic isotope effects with 4,4-dideuterio-1,4-dihydronicotinamides, suggesting that the hydrogen-transfer step is solely rate limiting.This reaction is characterized by an intrinsic primary kinetic isotope effect of 1.3 and a very product-like transition state.The enzymatic reaction has been studied by determining the second-order rate constants for the reduction of the imino acid by the enzyme-NADH complex with 4,4-dideuterio and stereospecifically labeled 4-deuterio NADH.The primary isotope effect when the in-place hydrogen is protium is 3.80, and the secondary isotope effect when the in-flight hydrogen is protium is 1.21.Deuteration at one site lowers the isotope effects at the other by 13percent.The following conclusions emerge for the reduction of the imino acid by the enzyme-NADH complex: (1) the hydrogen-transfer step is at least rate contributing, (2) the transition state for this reaction is more symmetric than that of the nonenzymatic reaction, (3) both the C-4 hydrogens of NADH participate in the reaction coordinate motion, and (4) there is some nuclear tunneling in the reaction coordinate.The kinetic isotope effect for the oxidation of proline and proline-2-d by enzyme-NADP(+) is 4.1.
- Srinivasan, R.,Fisher, Harvey F.
-
p. 4301 - 4305
(2007/10/02)
-
- The Pyridinium-Dihydropyridine System. Reduction Potentials and the Mechanism of Oxidation of 1,4-Dihydropyridines by a Shiff Base
-
As a model system for the glutamate dehydrogenase catalyzed reductive amination of α-ketoglutarate we have studied the reduction of a Shiff base, Δ1-pyrroline-2-carboxylic acid, by a series of 14 N-1 and C-3 substituted 1,4-dihydropyridines, including NMNH, NADH, and NADPH.The reversible electrode potentials of eight of the dihydropyridines, all dihydronicotinamides, have also been determined.The reduction reaction has the following characteristics: (a) it is first order in protonated Schiff base (zwitterionic form) and first order in the dihydropyridine, (b) there is a small deuterium isotope effect when the C-4 position of the dihydropyridine is deuterated (1.20-1.57 at 25 deg C), (c) there is a direct transfer of hydrogen from C-4 of the dihydropyridine to C-2 of the pyrroline, (d) the rates for seven N-1 substituted dihydronicotinamides are correlated satisfactorily with ?* giving ρ* = -1.98 (H2O) and -1.79 (aqueous methanol), there being only a modest difference in rates in these two solvents, (e) there is a good correlation between the rates of reduction by the dihydronicotinamides and the E0 values of the reversible two-electron dihydropyridine-pyridinium couple, the effect being 31.0 mV per logarithmic unit of rate, (f) there is a close correlation between the rates of reduction of pyrroline and of flavin by the dihydropyridines, and (g) the enthalpy and entropy of activation for the rate-controlling step in the reduction by 1-benzyl-1,4-dihydronicotinamide are 15.7 kcal mol-1 and -7.6 eu.We believe that direct hydride transfer has taken place to produce proline in a single step and it can be inferred that the transition state closely resembles products in structure.The similarity between pyrroline and flavin reduction suggests that the latter reaction may also be a direct hydride transfer.
- Srinivasan, R.,Medary, Richard T.,Fisher, Harvey F.,Norris, Donald J.,Stewart, Ross
-
p. 807 - 812
(2007/10/02)
-