On the basis of the X-ray crystal structure of
scytalone dehydratase complexed 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 an anti elimination mechanism) were found to be important to catalysis or substrate binding. Steady-state kinetic parameters for the mutants support the native roles for the residues (Asn131, Asp31, His85, His110, Ser129, Tyr30, and Tyr50) within a syn elimination mechanism. Relative substrate specificities for the two physiological substrates,
scytalone and veremelone, versus a Ser129 mutant help assign the orientation of the substrates within the active site. His85Asn was the most damaging mutation to catalysis consistent with its native roles as a general base and a general
acid in a syn elimination. The additive effect of Tyr30Phe and Tyr50Phe mutations in the double mutant is consistent with their roles in protonating the substrate's carbonyl through a water molecule. Studies on a synthetic substrate, which has an anomeric
carbon atom which can better stabilize a carbocation than the physiological substrate (
vermelone), suggest that His110Asn prefers this substrate over
vermelone in order to balance the mutation-imposed weakness in promoting the elimination of
hydroxide from substrates. All mutant
enzymes bound a potent active-site inhibitor in near 1:1 stoichiometry, thereby supporting their active-site integrity. An X-ray crystal structure of the Tyr50Phe mutant indicated that both active-site waters were retained, likely accounting for its residual catalytic activity. Steady-state kinetic parameters with deuterated
scytalone gave kinetic
isotope effects of 2.7 on kcat and 4.2 on kcat/Km, suggesting that steps after
dehydration partially limit kcat. Pre-steady-state measurements of a single-
enzyme turnover with
scytalone gave a rate that was 6-fold larger than kcat. kcat/Km with
scytalone has a pKa of 7.9 similar to the pKa value for the ionization of the substrate's C6 phenolic
hydroxyl, whereas kcat was unaffected by pH, indicating that the anionic form of
scytalone does not bind well to
enzyme. With an alternate substrate having a pKa above 11, kcat/Km had a pKa of 9.3 likely due to the ionization of Tyr50. The non-
enzyme-catalyzed rate of
dehydration of
scytalone was nearly a billion-fold slower than the
enzyme-catalyzed rate at pH 7.0 and 25 degrees C. The non-
enzyme-catalyzed rate of
dehydration of
scytalone had a
deuterium kinetic
isotope effect of 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 rate. Thus,
scytalone dehydrates through an E1cb mechanism off the
enzyme.