Biochemistry - The Chemical Reactions of Living Cells, Volume 2 - D. Metzler 1980

Coenzymes are specialized natural reagents of a unique kind.
Pyridine nucleotide coenzymes and dehydrogenases
Direct hydrogen atom transfer

Although it was clear that the pyridine ring is reduced during The conversion of NAD⁺ to NADH, the exact position of hydrogen attachment remained unknown until 1944, when isotopic labels were first applied in biochemical research. When NAD⁺ was reduced with dithionite in ²Н₂O medium [Equation (6-65)], a ²Н atom was incorporated into the reduced pyridine ring. Chemical degradation of the ring demonstrated that this atom was located at position 4, i.e., para to the ring nitrogen [68] (Fig. 8-10). Later, Westheimer and coworkers showed that during the enzymatic reduction of NAD⁺ interacting with СН₃—С²Н₂ОН, one of the ²Н atoms [Equation (6-65)] is transferred to the resulting NADH, providing Evidence for the direct transfer of a hydrogen atom [69]. Furthermore, when the NAD²H obtained in this way was reoxidized enzymatically with acetaldehyde to regenerate NAD⁺ and ethanol, the ²Н label was completely removed.

This was one of the earliest Examples illustrating the ability of an enzyme to select between two identical atoms in a prochiral center (Chapter 6, Section D, 2). The two hydrogen atoms at position 4 of NADH were designated as Н_А (now called пpo-R) and НВ (пpo-S), and the two faces of the nicotinamide ring as A and B. Alcohol dehydrogenase invariably removes the Н_А (пpo-R) hydrogen atom. Malate, isocitrate, lactate, and D-glycerate dehydrogenases share this same hydrogen Specificity [70]. Meanwhile, dehydrogenases acting on glucose-6-phosphate, glutamate, 6-phosphogluconate, and glyceraldehyde 3-phosphate remove the пpo-S-hydrogen¹⁾:

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If a hydrogen atom is transferred by an enzyme from the 4-position of NADH or NADPH to an aldehyde or ketone to yield an alcohol, the Location OF THE hydrogen atom in the alcohol is also stereospecific. Thus, in the presence of alcohol dehydrogenase, NAD²H converts acetaldehyde into (R)-mono-[²Н]-ethanol [Equation (6-65)]. Similarly, Pyruvate is reduced by Lactate dehydrogenase to L-lactate, and so forth.

Numerous attempts have been made to determine whether the rate-limiting step for dehydrogenases involves The transfer of a hydride ion or a hydrogen atom. In one study [71], para-substituted benzaldehydes were reduced by NADH and NAD²H using Yeast alcohol dehydrogenase as a catalyst. This made it possible to apply the Hammett equation [Equation (3-66)] to the kinetic Data analysis. For a series of benzaldehydes with widely varying σ⁺ values, a ρ value of +2.2 was obtained for the rate constants with both NADH and NAD²H. Thus, electron-withdrawing substituents in the para position accelerate the reaction. Although the precise significance of this observation is not entirely clear [71], the relatively low ρ value is presumably incompatible with a mechanism requiring the complete transfer of a single electron from NADH to acetaldehyde in the initial reaction step. The primary isotope effect for the rate constants is 3.6, indicating that the C—Н bond in the NADH molecule is cleaved in the rate-determining step. The fact that the isotope effect remains constant for all substituted benzaldehydes strongly Supports the hydride-ion transfer mechanism [71].

¹⁾ For more detailed information on the stereospecificity of NAD-dependent dehydrogenases, the reader is referred to a recently published review by You K., Arnold L. J., Jr., Allison W. S., Kaplan N. О. (1978). Trends Biochem. Sсi., 3, 265. — Transl. note.