63879-95-8Relevant academic research and scientific papers
Chondroitin-4-O-sulfatase from Bacteroides thetaiotaomicron: Exploration of the substrate specificity
Malleron, Annie,Benjdia, Alhosna,Berteau, Olivier,Le Narvor, Christine
scheme or table, p. 96 - 99 (2012/07/01)
Bacterial sulfatases can be good tools to increase the molecular diversity of glycosaminoglycan synthetic fragments. A chondroitin 4-O-sulfatase from the human commensal bacterium Bacteroides thetaiotaomicron has recently been identified and expressed. In order to use this enzyme for synthetic purposes, the minimal structure required for its activity has been determined. For that, four 4-O-sulfated monosaccharides and one 4-O-sulfated disaccharide have been synthesized and used as substrates with the sulfatase. The minimum structure was shown to be a disaccharide but in contrast to the natural substrate, which must have a 4,5-insaturation, the enzyme accepts as substrate, a disaccharide with a saturated glucuronic acid at the non-reducing end and even a glucopyranosyl moiety without the carboxylic acid functionality.
Elucidation of the mechanism of polysaccharide cleavage by chondroitin AC lyase from Flavobacterium heparinum
Rye, Carl S.,Withers, Stephen G.
, p. 9756 - 9767 (2007/10/03)
Chondroitin AC lyase from Flavobacterium heparinum degrades chondroitin sulfate glycosaminoglycans via an elimination mechanism resulting in disaccharides or oligosaccharides with Δ4,5-unsaturated uronic 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 inhomogeneous nature of the polymeric substrates. The creation of a new class of synthetic substrates for this enzyme has allowed the measurement of defined and reproducible kcat and Km values and has expanded the range of mechanistic studies that can be performed. The primary deuterium kinetic isotope effect upon kcat/Km for the abstraction of the proton α to the carboxylic acid was measured to be 1.67 ± 0.07, showing that deprotonation occurs in a rate-limiting step. Using substrates with leaving groups of differing reactivity, a flat 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 with this was the measurement of a secondary deuterium kinetic isotope effect upon kcat/Km of 1.01 ± 0.03 on a 4-{2H}-substrate, indicating that no sp2 character is developed at C4 during the rate-limiting step, thereby ruling out a concerted syn-elimination.
