Elucidation of the Mechanism of Polysaccharide Cleavage by Chondroitin AC Lyase from Flavobacterium heparinum

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• 作者：Carl S. Rye and Stephen G. Withers
• 刊名：Journal of the American Chemical Society
• 出版年：2002
• 出版时间：August 21, 2002
• 年：2002
• 卷：124
• 期：33
• 页码：9756 - 9767
• 全文大小：255K
• 年卷期：v.124,no.33(August 21, 2002)
• ISSN：1520-5126

文摘

Chondroitin AC lyase from Flavobacterium heparinum degrades chondroitin sulfate glycosaminoglycans via an elimination mechanism resulting in disaccharides or oligosaccharides with s/gifchars/Delta.gif" BORDER=0 >4,5-unsaturateduronic acid residues at their nonreducing end. Mechanistic details concerning the ordering of the bond-breaking and -forming steps of this enzymatic reaction are nonexistent, mainly due to the inhomogeneousnature of the polymeric substrates. The creation of a new class of synthetic substrates for this enzyme hasallowed the measurement of defined and reproducible k_cat and K_m values and has expanded the range ofmechanistic studies that can be performed. The primary deuterium kinetic isotope effect upon k_cat/K_m forthe abstraction of the proton s/gifchars/alpha.gif" BORDER=0> to the carboxylic acid was measured to be 1.67 ± 0.07, showing thatdeprotonation occurs in a rate-limiting step. Using substrates with leaving groups of differing reactivity, aflat linear free energy relationship was produced, indicating that the C4-O4 bond is not broken in a rate-determining step. Taken together, these results strongly suggest a stepwise mechanism. Consistent withthis was the measurement of a secondary deuterium kinetic isotope effect upon k_cat/K_m of 1.01 ± 0.03 ona 4-{²H}-substrate, indicating that no sp² character is developed at C4 during the rate-limiting step, therebyruling out a concerted syn-elimination.