Formamides are aldehyde analogues that have demonstrated potent and selective inhibition of human alcohol dehydrogenase isoenzymes. The αα, γ2γ2, and σσ isoforms have all been found to be strongly inhibited by substituted formamides. In this paper, the structure of the αα isoform of human alcohol dehydrogenase complexed with N-cyclopentyl-N-cyclobutylformamide was determined by X-ray crystallography to 2.5 Å resolution, β1β 1 isoform of human alcohol dehydrogenase complexed with N-benzylformamide and with N-heptylformamide was determined to 1.6 and 1.65 Å resolution, respectively, and the structure of the γ 2γ2 isoform complexed with N-1-methylheptylformamide was determined to 1.45 Å resolution. These structures provide the first substrate-level view of the local structural differences that give rise to the individual substrate preferences shown by these highly related isoenzymes. Consistent with previous work, the carbonyl oxygen of the inhibitors interacts directly with the catalytic zinc and the hydroxyl group of Thr48 (Ser48 for γ2γ2) of the enzyme. The benzene ring of N-benzylformamide and the carbon chains of N-heptylformamide and N-1-methylheptylformamide interact with the sides of the hydrophobic substrate pocket whose size and shape is dictated by residue exchanges between the β1β1 and γ2γ2 isoenzymes. In particular, the exchange of Ser for Thr at position 48 and the exchange of Val for Leu at position 141 in the γ2γ 2 isoenzyme create an environment with stereoselectivity for the R-enantiomer of the branched N-1-methylheptylformamide inhibitor in this isoenzyme. The primary feature of the αα isoform is the Ala for Phe93 exchange that enlarges the active site near the catalytic zinc and creates the specificity for the branched N-cyclopentyl-N-cyclobutylformamide inhibitor, which shows the greatest selectivity for this unique isoenzyme of any of the formamide inhibitors.
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