A vibrational analysis of the catalytically important C4-H bonds of NADH bound to lactate or malate dehydrogenase: ground-state effects

Abstract

We have measured the frequency of the carbon-hydrogen stretching mode of the pro-R and pro-S C4-H bonds of NADH in solution and when bound to pig heart lactate (LDH) or mitochondrial malate (mMDH) dehydrogenases. This is achieved by specifically deuterating the C4 pro-R or pro-S hydrogens of NADH and determining the frequencies of the resulting C4-D stretches by Raman difference spectroscopy. We find that the frequencies of the two C4-D stretching modes for the two bonds are essentially the same for the unliganded coenzyme. On the other hand, the position of the pro-S-[4-2H]NADH stretch shifts upward by about 23-30 cm-1 in its binary complex with either lactate or malate dehydrogenase relative to that observed in solution, while that for the bound pro-R-[4-2H]NADH is relatively unchanged. The fact that the frequency of the pro-R hydrogen is not significantly affected during complex formation suggests that the rate enhancements for reaction of substrate with NADH brought about by both pig heart LDH and mMDH apparently do not involve either stabilization or destabilization of the pro-R hydrogen of NADH in enzyme-coenzyme binary complexes, in agreement with previous chemical studies. That these proteins are able to regulate the frequencies of the two C4-D bonds differentially, and hence the electronic distributions in these bonds, has important implications for the stereochemical reactions catalyzed by the NAD dehydrogenases, and this is discussed.(ABSTRACT TRUNCATED AT 250 WORDS)

Vibrational analysis of NADH cofactors bound to glycerol‐3‐phosphate dehydrogenase and dogfish lactate dehydrogenase

“…Such a shift (or the absence of it) has been observed in various enzymes studied previously. 8,9,14,16,21 The band at 1547 cm 1 in the solutions spectrum of NADH [ Fig. 3(b)] is due to C C and C O stretch combinations based on vibrational calculations using ab initio quantum mechanical methods.…”

“…In particular, while large shifts from solution positions are observed for some bands of the C C stretch character and the carboxamide C O stretch and NH 2 rock, for NADH bound to LDH, none are found for NADH bound to G3PDH. It is most likely that the nicotinamide ring, which can adopt a range of angles from half-boat to planar in solution, 14 freezes to a near-planar ring geometry when bound to G3PDH. This structural conclusion is corroborated by the observation that the wavenumbers of the two C-4-H stretches (pro-R and pro-S C-4-H) are nearly the same for NADH bound to G3PDH (data not reported).…”

Interpretation of V/K isotope effects for enzymatic reactions exhibiting multiple isotopically sensitive steps

“…Significant binding isotope effects have also been measured in the reaction catalysed by human brain hexokinase (Lewis and Schramm, 2003), lactate dehydrogenase (LaReau et al, 1989;Gawlita et al, 1995a, b), phosphoenolpyruvate carboxylase (Gawlita et al, 1995a, b), and pyruvate kinase (Gawlita et al, 1995a, b). Furthermore, a number of spectroscopic studies have demonstrated significant polarization of substrate carbonyls upon binding enzyme and prior to catalysis that would be expected to yield corresponding isotope effects on the binding steps (Belasco and Knowles, 1980;Kurz et al, 1985;Deng et al, 1989Deng et al, , 1992Tonge and Carey, 1992). Effects such as these also can be viewed as potential contributors to any measured kinetic isotope effect intended to evaluate the transition state during the step of actual covalent rearrangement.…”

Enzyme Active Site Interactions by Raman/FTIR, NMR, and Ab Initio Calculations

“…The intensity ratios of these two bands are approximately 2:1 in both cases. In order to understand these spectral changes in terms of nicotinamide structural changes quantitatively, we have performed normal mode analysis based on quantum mechanical ab initio and semiempirical calculations (Deng, Burgner, & Callender, 1992; Deng, Zheng, Sloan, Burgner, & Callender, 1992). …”

“…The orientation of the amide group affects the C4–D stretch frequency of the reduced nicotinamide ring. A rotation of the C=O bond from cisoid to transoid will raise both C4–D stretches by about 10–20 cm −1 (Deng, Zheng, Sloan, Burgner, & Callender, 1992). Hence, we expect that the average frequency of the two C4–D stretches will increase as NADH binds to LDH.…”