Stereochemistry of the Enolization of Scytalone by Scytalone Dehydratase

In D₂O, scytalone exchanges its two C2 hydrogen atoms for deuterium atoms at different rates. At pD 7.0 and 25 °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 D₂O and then back exchanging with H₂O) 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 °C), 5.1 (25 °C), and 6.7 (6.8 °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.