Elucidation of the mechanism of polysaccharide cleavage by chondroitin AC lyase from Flavobacterium heparinum

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.