An atypical approach identifies TYR234 as the key base catalyst in chondroitin AC lyase

Chembiochem : a European Journal of Chemical Biology
Carl S RyeStephen G Withers

Abstract

Chondroitin AC lyase from Flavobacterium heparinum catalyses the degradation of chondroitin by an anionic E1cb elimination mechanism that involves proton abstraction from C5 of glucuronic 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 kcat/Km=2.7x10(6) M(-1) s(-); this is very similar to that of the natural chondroitin substrate (kcat/Km=1.3x10(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 proton 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 identific...Continue Reading

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Citations

Aug 21, 2013·Accounts of Chemical Research·Seino A K Jongkees, Stephen G Withers
Sep 2, 2010·Glycobiology·Marie-Line Garron, Miroslaw Cygler
Oct 12, 2010·The Biochemical Journal·Vincent LombardBernard Henrissat
Feb 26, 2016·Biotechnology for Biofuels·Logan C MacDonaldBryan W Berger
Jan 31, 2014·The Journal of Biological Chemistry·David ParkSatish K Nair

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