In D(2)O,
scytalone exchanges its two C2
hydrogen atoms for
deuterium atoms at different rates. At pD 7.0 and 25 degrees C, half-lives for the exchanges are 0.8 and 10 days for the pro-S and pro-R hydrogens, respectively. The differential exchange rates allow for the preparation of multiple
scytalone samples (through incubation of
scytalone in D(2)O and then back exchanging with H(2)O) having differential levels of
deuterium enrichment at the C2 pro-S and pro-R positions. From these samples, the stereochemical preference for
hydrogen abstraction during the
dehydration reaction mediated by the
enzyme scytalone dehydratase was determined. At pH 7. 0,
deuterium at the pro-S position has little effect on
enzyme catalysis, whereas
deuterium at the pro-R position produces kinetic
isotope effects of 2.3 (25 degrees C), 5.1 (25 degrees C), and 6.7 (6.8 degrees C) on k(cat), k(cat)/K(m), and the single-turnover rate, respectively. The results are fully consistent with the
enzyme catalyzing a syn elimination through an E1cb-like mechanism. The syn elimination is compatible with the interactions realized between a
scytalone boat conformation and key active site residues as modeled from multiple X-ray crystal structures of the
enzyme in complexes with inhibitors.