Roles of Substrate Distortion and Intramolecular Hydrogen Bonding in Enzymatic Catalysis by Scytalone Dehydratase

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• 作者：Ya-Jun Zheng ; Gregory S. Basarab ; and Douglas B. Jordan
• 刊名：Biochemistry
• 出版年：2002
• 出版时间：January 22, 2002
• 年：2002
• 卷：41
• 期：3
• 页码：820 - 826
• 全文大小：85K
• 年卷期：v.41,no.3(January 22, 2002)
• ISSN：1520-4995

文摘

Alternative substrates and site-directed mutations of active-site residues are used to probefactors controlling the catalytic efficacy of scytalone dehydratase. In the E1cb-like, syn-elimination reactionscatalyzed, efficient catalysis requires distortion of the substrate ring system to facilitate proton abstractionfrom its C2 methylene and elimination of its C3 hydroxyl group. Theoretical calculations indicate thatsuch distortions are more readily achieved in the substrate 2,3-dihydro-2,5-dihydroxy-4H-benzopyran-4-one (DDBO) than in the physiological substrates vermelone and scytalone by ~2 kcal/mol. A survey of12 active-site amino acid residues reveals 4 site-directed mutants (H110N, N131A, F53A, and F53L)have higher relative values of k_cat and k_cat/K_m for DDBO over scytalone and for DDBO over vermelonethan the wild-type enzyme, thus suggesting substrate-distortion roles for the native residues in catalysis.A structural link for this function is in the modeled enzyme-substrate complex where F53 and H110 arepositioned above and below the substrate's C3 hydroxyl group, respectively, for pushing and pulling theleaving group into the axial orientation of a pseudo-boat conformation; N131 hydrogen-bonds to the C8hydroxyl group at the opposite end of the substrate, serving as a pivot for the actions of F53 and H110.Deshydroxyvermelone lacks the phenolic hydroxyl group and the intramolecular hydrogen bond ofvermelone. The relative values of k_cat (95) and k_cat/K_m (1800) for vermelone over deshydroxyvermelonefor the wild-type enzyme indicate the importance of the hydroxyl group for substrate recognition andcatalysis. Off the enzyme, the much slower rates for the solvolytic dehydration of deshydroxyvermeloneand vermelone are similar, thus specifying the importance of the hydroxyl group of vermelone for enzymecatalysis.