Catalytic Mechanism of Scytalone Dehydratase:  Site-Directed Mutagenisis, Kinetic Isotope Effects, and Alternate Substrates

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 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 with 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 enzyme. 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-catalyzed rate of dehydration of scytalone w