Metal ion activator effects on intrinsic isotope effects for hydride transfer from decarboxylation in the reaction catalyzed by the NAD-malic enzyme from Ascaris suum.

The mechanism of the oxidative decarboxylation reaction catalyzed by the NAD-malic enzyme from Ascaris suum has been examined with several different divalent metal ion activators and dinucleotide substrates. Primary deuterium and tritium isotope effects have been obtained and, in combination with the partitioning ratios of the oxalacetate intermediate to malate and pyruvate, have been used to calculate commitment factors, intrinsic deuterium isotope effects on the hydride transfer step, and intrinsic 13C isotope effects for the decarboxylation step. A survey of malate analogs has been undertaken to define the geometry of the active site and to identify functional groups on malate important for substrate binding. With NAD as dinucleotide substrate, a direct correlation between the size of the divalent metal ion activator and the intrinsic deuterium isotope effect is observed. An isotope effect significantly greater than the semiclassical limit is seen when Cd2+ is the metal ion activator, indicating a substantial tunneling contribution. The primary intrinsic 13C isotope effect on the decarboxylation step increases over the series Mg2+ < Mn2+ < Cd2+, which is in contrast to the equal isotope effects measured for these metal ions for the nonenzymatic decarboxylation of oxalacetate [Grissom, C. B., & Cleland, W. W. (1986) J. Am. Chem. Soc. 108, 5582]. With Mn2+ or Cd2+ as the divalent metal ion activator, the data support a stepwise mechanism for the enzymatic oxidative decarboxylation with NAD as the dinucleotide substrate, but a change to a concerted mechanism is indicated with more redox-positive dinucleotide substrates as suggested previously with Mg2+ as activator [Karsten, W. E., & Cook, P. F. (1994) Biochemistry 33, 2096].(ABSTRACT TRUNCATED AT 250 WORDS)

A 13C isotopomer n.m.r. method for monitoring incomplete beta-oxidation of fatty acids in intact tissue.

An n.m.r. method is presented for monitoring the extent to which fatty acids undergo beta-oxidation without release of shorter-chain intermediates. It is based upon a 13C isotopomer analysis of glutamate from tissue presented with a mixture of [2,4,6,8-13C]octanoate and [1,2,3,4-13C]octanoate. The method does not require steady-state metabolic or isotopic conditions, so it may be applied during a variety of metabolic circumstances, including perfused tissue under stress and in vivo. We have tested the method in perfused rat hearts during anoxia, a model where previous work has shown that beta-oxidation of palmitate is incomplete and shorter-chain intermediates are released [Rabinowitz and Hercker (1974) Arch. Biochem. Biophys. 161, 621-627]. Indeed, n.m.r. spectra of freeze-clamped, acid-extracted tissue show that octanoate undergoes complete beta-oxidation in control normoxic rat hearts, but not in anoxic hearts. Complete beta-oxidation of octanoate was observed under a number of other metabolic conditions in perfused rat hearts, including low-pressure-induced ischaemia, KCl arrest and in the presence of high concentrations of competing substrates. We also demonstrate that the technique is applicable in intact tissue by taking direct measurements in perfused rat hearts using a recently published [13C]homonuclear decoupling technique and in in vivo heart and liver removed from rats after an intravenous infusion of a mixture of [2,4,6,8-13C]octanoate and [1,2,3,4-13C]octanoate.

Multiple isotope effects with alternative dinucleotide substrates as a probe of the malic enzyme reaction.

Deuterium isotope effects and 13C isotope effects with deuterium- and protium-labeled malate have been obtained for both NAD- and NADP-malic enzymes by using a variety of alternative dinucleotide substrates. With nicotinamide-containing dinucleotides as the oxidizing substrate, the 13C effect decreases when deuterated malate is the substrate compared to the value obtained with protium-labeled malate. These data are consistent with a stepwise chemical mechanism in which hydride transfer precedes decarboxylation of the oxalacetate intermediate as previously proposed [Hermes, J. D., Roeske, C. A., O'Leary, M. H., & Cleland, W. W. (1982) Biochemistry 21, 5106]. When dinucleotide substrates such as thio-NAD, 3-acetylpyridine adenine dinucleotide, and 3-pyridinealdehyde adenine dinucleotide that contain modified nicotinamide rings are used, the 13C effect increases when deuterated malate is the substrate compared to the value obtained with protium-labeled malate. These data, at face value, are consistent with a change in mechanism from stepwise to concerted for the oxidative decarboxylation portion of the mechanism. However, the increase in the deuterium isotope effect from 1.5 to 3 with a concomitant decrease in the 13C isotope effect from 1.034 to 1.003 as the dinucleotide substrate is changed suggests that the reaction may still be stepwise with the non-nicotinamide dinucleotides. A more likely explanation is that a beta-secondary 13C isotope effect accompanies hydride transfer as a result of hyperconjugation of the beta-carboxyl of malate as the transition state for the hydride transfer step is approached.(ABSTRACT TRUNCATED AT 250 WORDS)

Modification of a thiol at the active site of the Ascaris suum NAD-malic enzyme results in changes in the rate-determining steps for oxidative decarboxylation of L-malate.

A thiol group at the malate-binding site of the NAD-malic enzyme from Ascaris suum has been modified to thiocyanate. The modified enzyme generally exhibits slight increases in KNAD and Ki metal and decreases in Vmax as the metal size increases from Mg2+ to Mn2+ to Cd2+, indicative of crowding in the site. The Kmalate value increases 10- to 30-fold, suggesting that malate does not bind optimally to the modified enzyme. Deuterium isotope effects on V and V/Kmalate increase with all three metal ions compared to the native enzyme concomitant with a decrease in the 13C isotope effect, suggesting a switch in the rate limitation of the hydride transfer and decarboxylation steps with hydride transfer becoming more rate limiting. The 13C effect decreases only slightly when obtained with deuterated malate, suggestive of the presence of a secondary 13C effect in the hydride transfer step, similar to data obtained with non-nicotinamide-containing dinucleotide substrates for the native enzyme (see the preceding paper in this issue). The native enzyme is inactivated in a time-dependent manner by Cd2+. This inactivation occurs whether the enzyme alone is present or whether the enzyme is turning over with Cd2+ as the divalent metal activator. Upon inactivation, only Cd2+ ions are bound at high stoichiometry to the enzyme, which eventually becomes denatured. Conversion of the active-site thiol to thiocyanate makes it more difficult to inactivate the enzyme by treatment with Cd2+.

The kinetic mechanism of human placental aldose reductase and aldehyde reductase II.

The kinetic mechanism of NADPH-dependent aldehyde reductase II and aldose reductase, purified from human placenta, has been studied using L-glucuronate and DL-glyceraldehyde as their respective substrates. For aldehyde reductase II, the initial velocity and product inhibition studies (using NADP and gulonate) indicate that the enzyme reaction sequence is ordered with NADPH binding to the free enzyme and NADP being the last product to be released. Inhibition patterns using menadione (an analog of the aldehydic substrate) and ATP-ribose (an analog of NADPH) are also consistent with a compulsory ordered reaction sequence. Isotope effects of deuterium-substituted NADPH (NADPD) also corroborate the above reaction scheme and indicate that hydride transfer is not the sole rate-limiting step in the reaction sequence. For aldose reductase, initial velocity patterns, product, and dead-end inhibition studies indicate a random binding pattern of the substrates and an ordered release of product; the coenzyme is released last. A steady-state random mechanism is also consistent with deuterium isotope effects of NADPD on the reaction sequence catalyzed by this enzyme. However, the hydride transfer step seems to be more rate determining for aldose reductase than for aldehyde reductase II.