|Title||An atypical approach identifies TYR234 as the key base catalyst in chondroitin AC lyase|
|Publication Type||Journal Article|
|Year of Publication||2006|
|Authors||Rye, CS, Matte, A, Cygler, M, Withers, SG|
Chondroitin AC lyase from Flavobacterium heparinum catalyses the degradation of chondroitin by on anionic E1cb elimination mechanism that involves proton abstraction from C5 of glucuroni. c acid. The lyase also carries out efficient proton transfer to a sugar nitronate anion, which was designed originally as an inhibitor of the enzyme, with a second-order rate constant of k(cat)/k(m) = 2.7 x 10(6) M-1 S-; this is very similar to that of the natural chon1 droitin substrate (k(cat)/k(m) = 1.3 x 10(6) M-1 S-1). Studies with this nitronate should therefore provide insight into the proton-transfer step (general base catalysis) within this mechanism. Indeed, the Tyr234Phe mutant of the enzyme was essentially inactive with the natural substrate and correspondingly did not catalyse wproton transfer to the nitronate, thereby implicating this residue as the general base catalyst. Parallel studies designed to identify the acid catalyst were carried out by using a substrate with a 2,4-dinitrophenol leaving group that needs no acid assistance for departure. These results are consistent with Tyr234 also playing the role of acid catalyst. Not only do these studies confirm the suspected role of Tyr234, but also they validate a new methodology for identification of acid/base catalysts in lyases and epimerases of this type. In addition a structural and mechanistic rationale is provided for different active-site acid/base configurations in syn and anti lyases.