Alternative substrates and site-directed mutations of active-site residues are used to probe factors controlling the catalytic efficacy of
scytalone dehydratase. In the E1cb-like, syn-elimination reactions catalyzed, efficient catalysis requires distortion of the substrate ring system to facilitate
proton abstraction from its C2 methylene and elimination of its C3
hydroxyl group. Theoretical calculations indicate that such distortions are more readily achieved in the substrate
2,3-dihydro-2,5-dihydroxy-4H-benzopyran-4-one (
DDBO) than in the physiological substrates
vermelone and
scytalone by approximately 2 kcal/mol. A survey of 12 active-site
amino acid residues reveals 4 site-directed mutants (H110N, N131A, F53A, and F53L) have higher relative values of k(cat) and k(cat)/K(m) for
DDBO over
scytalone and for
DDBO over
vermelone than the wild-type
enzyme, thus suggesting substrate-distortion roles for the native residues in catalysis. A structural link for this function is in the modeled
enzyme-substrate complex where F53 and H110 are positioned above and below the substrate's C3
hydroxyl group, respectively, for pushing and pulling the leaving group into the axial orientation of a pseudo-boat conformation; N131 hydrogen-bonds to the C8
hydroxyl group at the opposite end of the substrate, serving as a pivot for the actions of F53 and H110. Deshydroxyvermelone lacks the phenolic
hydroxyl group and the intramolecular hydrogen bond of
vermelone. The relative values of k(cat) (95) and k(cat)/K(m) (1800) for
vermelone over deshydroxyvermelone for the wild-type
enzyme indicate the importance of the
hydroxyl group for substrate recognition and catalysis. Off the
enzyme, the much slower rates for the solvolytic
dehydration of deshydroxyvermelone and
vermelone are similar, thus specifying the importance of the
hydroxyl group of
vermelone for
enzyme catalysis.