| 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 non-reducing end. A general mechanism for all polysaccharide lyases has been proposed, however no mechanistic details exist, mainly due to the inhomogeneous nature of the polymeric substrates available, which renders impossible the collection of reliable and reproducible data. Thus, three different types of substrate were developed that can be monitored by three different techniques: UV/Vis spectroscopy, fluorescence spectroscopy, and by the use of a fluoride ion-selective electrode. These synthetic substrates have allowed the measurement of defined and reproducible kinetic parameters previously unavailable using the polymeric natural substrates.; The creation of this new class of structurally defined substrates has allowed their modification in order to carry out a variety of mechanistic analyses aimed at deciphering the exact catalytic mechanism of chondroitin AC lyase. A combination of primary and secondary deuterium kinetic isotope effects together with a linear free energy relationship has revealed the details regarding the order of the bond breaking and making steps of the enzymatic reaction. These mechanistic studies have revealed the reaction mechanism to elimination of the C4-linked leaving group. In addition, a solvent isotope exchange experiment has revealed that the exchange of deuterium at C5 does not compete with the departure of the leaving group, thus classifying the elimination mechanism as (E1cb)irr or (E1cb)I in which Tight binding inhibitors, in conjunction with X-ray crystallography, may allow the identification of key catalytic residues responsible for binding and catalysis. There are no known specific inhibitors of polysaccharide lyases reported in the literature, prompting investigation into this area. Two potential inhibitors of chondroitin AC lyase were synthesized, based on two different design concepts. The first, a disaccharide molecule containing a pre-formed C4-C5 alkene moiety in combination with an intact hexosamine leaving group sugar residue, surprisingly showed no inhibition. The second potential inhibitor was a novel 5-nitro sugar, designed to mimic the aci-carboxylate intermediate along the reaction pathway. This compound was found to be a competitive inhibitor of the enzyme with a Ki value of ∼0.7 mM. Unfortunately this relatively large inhibition constant is not representative of a transition state analogue, which would be expected to bind much tighter to the enzyme. |
