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

• Published in: Journal of the American Chemical Society Vol. 124; no. 33; pp. 9756 - 9767
• Main Authors: RYE, Carl S., WITHERS, Stephen G.
• Format: Journal Article
• Language: English
• Published: WASHINGTON American Chemical Society 21.08.2002; Amer Chemical Soc
• Subjects: Analytical chemistry; Catalysis; Chemistry; Chemistry, Multidisciplinary; Chondroitin Lyases - antagonists & inhibitors; Chondroitin Lyases - chemistry; Chondroitin Lyases - metabolism; Chondroitin Sulfates - chemistry; Chondroitin Sulfates - metabolism; Deuterium; Exact sciences and technology; Flavobacterium - enzymology; Hydrocarbons, Fluorinated - chemical synthesis; Hydrocarbons, Fluorinated - chemistry; Hydrocarbons, Fluorinated - metabolism; Hydrogen-Ion Concentration; Kinetics; Miscellaneous; Nuclear Magnetic Resonance, Biomolecular; Physical Sciences; Polysaccharides - chemical synthesis; Polysaccharides - chemistry; Polysaccharides - metabolism; Science & Technology; Uronic Acids - chemical synthesis; Uronic Acids - chemistry; Uronic Acids - metabolism; EXPLAIN; ACTIVE-SITE; CRYSTAL-STRUCTURE; UNEXPECTED ACIDITY; BETA-ELIMINATION; DEGRADATION; CATALYZED PROTON ABSTRACTION; DERIVATIVES; CARBON ACIDS; ACTION PATTERN; Polysaccharide-lyases; Chemistry; Flavobacteriaceae; Enzyme; Bacteria; Flavobacterium heparinum; Lyases; Carbon-oxygen lyases; Polysaccharide; Chondroitin AC lyase
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja020627c

Chondroitin AC lyase from Flavobacterium heparinum degrades chondroitin sulfate glycosaminoglycans via an elimination mechanism resulting in disaccharides or oligosaccharides with Delta4,5-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 k(cat) and K-m values and has expanded the range of mechanistic studies that can be performed. The primary deuterium kinetic isotope effect upon k(cat)/K-m for the abstraction of the proton a 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 k(cat)/K-m of 1.01 +/- 0.03 on a 4-{H-2}-substrate, indicating that no sp(2) character is developed at C4 during the rate-limiting step, thereby ruling out a concerted syn-elimination.