Alternative binding modes in abortive NADH-alcohol complexes of horse liver alcohol dehydrogenase.

Bryce V Plapp, Ramaswamy Subramanian

Archives of biochemistry and biophysics 2021 Apr 15

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Enzymes typically have high specificity for their substrates, but the structures of substrates and products differ, and multiple modes of binding are observed. In this study, high resolution X-ray crystallography of complexes with NADH and alcohols show alternative modes of binding in the active site. Enzyme crystallized with the good substrates NAD+ and 4-methylbenzyl alcohol was found to be an abortive complex of NADH with 4-methylbenzyl alcohol rotated to a "non-productive" mode as compared to the structures that resemble reactive Michaelis complexes with NAD+ and 2,2,2-trifluoroethanol or 2,3,4,5,6-pentafluorobenzyl alcohol. The NADH is formed by reduction of the NAD+ with the alcohol during the crystallization. The same structure was also formed by directly crystallizing the enzyme with NADH and 4-methylbenzyl alcohol. Crystals prepared with NAD+ and 4-bromobenzyl alcohol also form the abortive complex with NADH. Surprisingly, crystals prepared with NAD+ and the strong inhibitor 1H,1H-heptafluorobutanol also had NADH, and the alcohol was bound in two different conformations that illustrate binding flexibility. Oxidation of 2-methyl-2,4-pentanediol during the crystallization apparently led to reduction of the NAD+. Kinetic studies show that high concentrations of alcohols can bind to the enzyme-NADH complex and activate or inhibit the enzyme. Together with previous studies on complexes with NADH and formamide analogues of the carbonyl substrates, models for the Michaelis complexes with NAD+-alcohol and NADH-aldehyde are proposed. Copyright © 2021 Elsevier Inc. All rights reserved.

Citation

Bryce V Plapp, Ramaswamy Subramanian. Alternative binding modes in abortive NADH-alcohol complexes of horse liver alcohol dehydrogenase. Archives of biochemistry and biophysics. 2021 Apr 15;701:108825