Interaction between dehydrogenases and a new NAD -isomer.

Area of Science:

• Biochemistry
• Enzymology
• Organic Chemistry

Background:

• Nicotinamide-adenine-dinucleotide (NAD) is a crucial coenzyme in redox reactions.
• Enzyme kinetics and coenzyme binding are fundamental to understanding metabolic pathways.
• Investigating coenzyme analogs can reveal insights into enzyme mechanisms.

Purpose of the Study:

• To synthesize and characterize a novel NAD isomer, (D,L)-NAD, where L-ribose replaces D-ribose.
• To compare the binding affinities and kinetic parameters of (D,L)-NAD and its dihydroform, (D,L)-NADH, with those of native NAD and NADH.
• To evaluate the enzymatic activity of (D,L)-NAD and (D,L)-NADH with lactate dehydrogenase and alcohol dehydrogenase.

Main Methods:

• Chemical synthesis of the L-ribose substituted NAD isomer.
• Enzyme kinetic assays using lactate dehydrogenase and alcohol dehydrogenase from horse liver and yeast.
• Determination of Michaelis constants (Km) and catalytic constants (V/Et).

Main Results:

• The synthesized coenzyme isomer, (D,L)-NAD, and its dihydroform, (D,L)-NADH, exhibit significantly lower binding affinities to lactate dehydrogenase and alcohol dehydrogenase compared to NAD and NADH.
• (D,L)-NAD and (D,L)-NADH function as hydrogen acceptor and donor, respectively, in enzymatic reactions.
• Michaelis constants were consistently increased for the NAD isomer compared to the native coenzyme.
• Catalytic constants (V/Et) were generally decreased, with an exception for (D,L)-NADH with alcohol dehydrogenase from horse liver.

Conclusions:

• The stereochemistry of the ribose moiety in NAD significantly influences coenzyme binding and catalytic efficiency.
• (D,L)-NAD and (D,L)-NADH serve as valuable tools for probing the active sites and mechanisms of NAD-dependent dehydrogenases.
• Enzyme engineering and drug design could potentially leverage these findings to modulate enzyme activity.