@article { ISI:000177455000040,
	title = {Elucidation of the mechanism of polysaccharide cleavage by chondroitin AC lyase from Flavobacterium heparinum},
	journal = {JOURNAL OF THE AMERICAN CHEMICAL SOCIETY},
	volume = {124},
	number = {33},
	year = {2002},
	month = {AUG 21},
	pages = {9756-9767},
	abstract = {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.},
	issn = {0002-7863},
	doi = {10.1021/ja020627c},
	author = {Rye, CS and Withers, SG}
}
