Tight binding inhibitors of scytalone dehydratase: Effects of site-directed mutations

We explore the use of site-directed mutations of scytalone dehydratase to s tudy inhibitor binding interactions. The enzyme is the physiological target of new fungicides and the subject of inhibitor design and optimization. X- ray structures show that potent inhibitors (K-i's approximate to 10(-11) M) interact mostly with II amino acid side chains and, in some cases, with a single backbone amide. Fifteen site-directed mutants of the 11 enzyme resid ues were prepared to disrupt enzyme-inhibitor interactions. and inhibition constants for 13 inhibitors were determined to assess changes in binding po tencies. The results indicate that two of the six hydrogen bonds (always pr esent in X-ray structures of native enzyme-inhibitor complexes) are not imp ortant for inhibitor binding. The other four hydrogen bonds are important f or inhibitor binding, and the strength of the individual bonds is inhibitor -dependent. Inhibitor atoms remote from the hydrogen bonds influence their strength, presumably by effecting small changes in inhibitor orientation. S everal hydrophobic amino acid residues are important recognition elements f or lipophilic inhibitor functionalities, which is fully consistent with X-r ay structures determined from crystals of enzyme-inhibitor complexes grown at neutral pH but not with those determined from crystals grown under acidi c conditions. This study of mutant enzymes complements insights from X-ray structures and structure-activity relationships of the wild-type enzyme for refining Views of inhibitor recognition.