Vibrational structure of NAD(P) cofactors bound to three NAD(P) dependent enzymes

An investigation of ground state activation

Yong Qing Chen, Jeroen Van Beek, Hua Deng, John Burgner, Robert Callender

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

The NAD(P) dependent dehydrogenases and reductases stereospecifically catalyze the transfer of a hydride ion from C4 of the dihydronicotinamide of the NAD(P) ring to the substrate. We have investigated the vibrational structure of the important C4-H coordinate for NAD(P)H and NADP+ bound to three enzymes in binary and ternary (Michaelis mimics) complexes: the A-side specific lactate dehydrogenase (LDH) and dihydrofolate reductase (DHFR) and the B-side specific glycerol-3-phosphate dehydrogenase (G3PDH). This is achieved by specifically deuterating the C4 pro-R or pro-S hydrogens of the reduced ring or the C4 hydrogen of the oxidized ring, which results in a vibrational mode localized to the stretching motion of the labeled C4-H bond. We observed relatively minor changes in the stretch frequencies of the C4-H bonds showing that the electronic nature of the bond is not substantially modified by cofactor binding, a mechanism previously proposed to be involved in enzymic "activation" toward catalysis. However, from the observed band narrowing of the C4-D stretch band, it is clear that interactions at the active site in all three proteins greatly reduced the conformational flexibility of either the reduced or oxidized ring as the cofactor moves from solution to the binary complex or ternary complex, guiding the ring structure from the ensemble of structures accessible in solution toward a selected set. Moreover, as NAD(P)H binds to LDH or DHFR forming binary as well as ternary Michaelis mimic complexes, the pro-R hydrogen is brought to a pseudoaxial orientation, which is thought to be proper geometry for the transition state of hydride transfer. Hence, ground state structural distortions imposed on the cofactor appear to populate preferentially the correct ring geometry for enzymic activity. Surprisingly, the mimics of their Michaelis complexes also contain a substantial second, presumably unproductive, population of the bound cofactor whereby the pro-S hydrogen is pseudoaxial. Unexpectedly, the geometry of NADH bound to G3PDH is nearly planar with the pro-R hydrogen slightly pseudoaxial. This would seem to be a poorly bound cofactor for catalysis although it may well be true that the transition state geometry for G3PDH is not that of LDH. How the results bear on various proposals concerning ground-state regulation of reactivity is discussed.

Original languageEnglish (US)
Pages (from-to)10733-10740
Number of pages8
JournalJournal of Physical Chemistry B
Volume106
Issue number41
DOIs
StatePublished - Oct 17 2002

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dehydrogenases
NAD
Ground state
enzymes
Hydrogen
Glycerolphosphate Dehydrogenase
Enzymes
Chemical activation
activation
ground state
L-Lactate Dehydrogenase
lactates
Tetrahydrofolate Dehydrogenase
Geometry
glycerols
rings
Hydrides
hydrogen
Catalysis
phosphates

ASJC Scopus subject areas

  • Physical and Theoretical Chemistry

Cite this

Vibrational structure of NAD(P) cofactors bound to three NAD(P) dependent enzymes : An investigation of ground state activation. / Chen, Yong Qing; Van Beek, Jeroen; Deng, Hua; Burgner, John; Callender, Robert.

In: Journal of Physical Chemistry B, Vol. 106, No. 41, 17.10.2002, p. 10733-10740.

Research output: Contribution to journalArticle

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N2 - The NAD(P) dependent dehydrogenases and reductases stereospecifically catalyze the transfer of a hydride ion from C4 of the dihydronicotinamide of the NAD(P) ring to the substrate. We have investigated the vibrational structure of the important C4-H coordinate for NAD(P)H and NADP+ bound to three enzymes in binary and ternary (Michaelis mimics) complexes: the A-side specific lactate dehydrogenase (LDH) and dihydrofolate reductase (DHFR) and the B-side specific glycerol-3-phosphate dehydrogenase (G3PDH). This is achieved by specifically deuterating the C4 pro-R or pro-S hydrogens of the reduced ring or the C4 hydrogen of the oxidized ring, which results in a vibrational mode localized to the stretching motion of the labeled C4-H bond. We observed relatively minor changes in the stretch frequencies of the C4-H bonds showing that the electronic nature of the bond is not substantially modified by cofactor binding, a mechanism previously proposed to be involved in enzymic "activation" toward catalysis. However, from the observed band narrowing of the C4-D stretch band, it is clear that interactions at the active site in all three proteins greatly reduced the conformational flexibility of either the reduced or oxidized ring as the cofactor moves from solution to the binary complex or ternary complex, guiding the ring structure from the ensemble of structures accessible in solution toward a selected set. Moreover, as NAD(P)H binds to LDH or DHFR forming binary as well as ternary Michaelis mimic complexes, the pro-R hydrogen is brought to a pseudoaxial orientation, which is thought to be proper geometry for the transition state of hydride transfer. Hence, ground state structural distortions imposed on the cofactor appear to populate preferentially the correct ring geometry for enzymic activity. Surprisingly, the mimics of their Michaelis complexes also contain a substantial second, presumably unproductive, population of the bound cofactor whereby the pro-S hydrogen is pseudoaxial. Unexpectedly, the geometry of NADH bound to G3PDH is nearly planar with the pro-R hydrogen slightly pseudoaxial. This would seem to be a poorly bound cofactor for catalysis although it may well be true that the transition state geometry for G3PDH is not that of LDH. How the results bear on various proposals concerning ground-state regulation of reactivity is discussed.

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