Kinetics and docking studies on the effect of chemical modification of NADH for redox reactions with dehydrogenases

Cofactor analogs promise important applications in biosynthesis. The effect of chemical modification on the reactivity of NADH for redox reactions catalyzed by dehydrogenases was examined in this work. Compared with the native NADH, kinetics and molecular docking studies with 8-(6-aminohexyl)-amino-NADH showed that its binding with alcohol dehydrogenase (ADH) was not much affected or even enhanced by a factor of 4.9-fold with lactate dehydrogenase (LDH), but complicated the binding of substrates to the enzymes. For ADH, the Michaelis constant for acetaldehyde decreased from 0.47 to 0.048 mM, while that of sodium pyruvate with LDH increased to 0.81 from 0.18 mM. On the other hand, the modified coenzyme showed a 19.3-fold decrease in turnover number (kcat) with ADH, while a slight increase with LDH. Molecular docking analysis showed that the hexanediamine arm on the modified coenzyme generated an extra hydrogen bond at the active site of ADH, as well as additional hydrophobic interactions with both ADH and LDH. It appeared that the apparently decreased reactivity of modified cofactor with ADH was caused mainly by the enhanced stability of ternary coenzyme–enzyme–substrate complex, while in the case of LDH, the reduced substrate binding as a result of the chemical modification of NADH led to a slight increase in the overall reaction reactivity.

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► Binding of 8-(6-aminohexyl)-amino-NADH with alcohol dehydrogenase (ADH) was not much affected.
► Binding of modified NADH with lactate dehydrogenase (LDH) was 4.9-times enhanced.
► Hexanediamine arm on modified NADH generated extra hydrogen bond and hydrophobic interactions with ADH and LDH.
► Decreased reactivity of modified NADH with ADH was caused by the enhanced stability of ternary coenzyme–enzyme–substrate complex.
► The reduced substrate binding as a result of the chemical modification of NADH led to a slight increase in the overall reaction reactivity in the case of LDH.