Catalytic mechanism of scytalone dehydratase: Site-directed mutagenesis, kinetic isotope effects, and alternate substrates

On the basis of the X-ray crystal structure of scytalone dehydratase comple xed with an active center inhibitor [Lundqvist, T., Rice, J., Hodge, C. N., Basarab, G. S., Pierce, J, and Lindqvist, Y. (1994) Structure (London) 2, 937-944], eight active-site residues were mutated to examine their roles in the catalytic mechanism. All but one residue (Lys73, a potential base in a n anti elimination mechanism) were found to be important to catalysis or su bstrate binding. Steady-state kinetic parameters for the mutants support th e native roles for the residues (Asn131, Asp31, His85, His110, Ser129, Tyr3 0, and Tyr50) within a syn elimination mechanism. Relative substrate specif icities for the two physiological substrates, scytalone and veremelone, ver sus a Ser129 mutant help assign the orientation of the substrates within th e active site. His85Asn was the most damaging mutation to catalysis consist ent with its native roles as a general base and a general acid in a syn eli mination. The additive effect of Tyr30Phe and Tyr50Phe mutations in the dou ble mutant is consistent with their roles in protonating the substrate's ca rbonyl through a water molecule. Studies on a synthetic substrate, which ha s an anomeric carbon atom which can better stabilize a carbocation than the physiological substrate (vermelone), suggest that His110Asn prefers this s ubstrate over vermelone in order to balance the mutation-imposed weakness i n promoting the elimination of hydroxide from substrates. All mutant enzyme s bound a potent active-site inhibitor in near 1:1 stoichiometry, thereby s upporting their active-site integrity. An X-ray crystal structure of the Ty r50Phe mutant indicated that both active-site waters were retained, likely accounting for its residual catalytic activity. Steady-state kinetic parame ters with deuterated scytalone gave kinetic isotope effects of 2.7 on k(cat ) and 4.2 on k(cat)/K-m, suggesting that steps after dehydration partially limit k(cat). Pre-steady-state measurements of a single-enzyme turnover wit h scytalone gave a rate that was 6-fold larger than k(cat). k(cat)/K-m with scytalone has a pK(a) of 7.9 similar to the pK(a) value for the ionization of the substrate's C6 phenolic hydroxyl, whereas k(cat) was unaffected by pH, indicating that the anionic form of scytalone does not bind well to enz yme. With an alternate substrate having a pK(a) above 11, k(cat)/K-m had a pK(a) of 9.3 likely due to the ionization of Tyr50. The non-enzyme-catalyze d rate of dehydration of scytalone was nearly a billion-fold slower than th e enzyme-catalyzed rate at pH 7.0 and 25 degrees C, The non-enzyme-catalyze d rate of dehydration of scytalone had a deuterium kinetic isotope effect o f 1.2 at pH 7.0 and 25 degrees C, and scytalone incorporated deuterium from D2O in the C2 position about 70-fold more rapidly than the dehydration rat e. Thus, scytalone dehydrates through an E1cb mechanism off the enzyme.