Document Detail

An atypical approach identifies TYR234 as the key base catalyst in chondroitin AC lyase.
MedLine Citation:
PMID:  16521140     Owner:  NLM     Status:  MEDLINE    
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 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.
Carl S Rye; Allan Matte; Miroslaw Cygler; Stephen G Withers
Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Chembiochem : a European journal of chemical biology     Volume:  7     ISSN:  1439-4227     ISO Abbreviation:  Chembiochem     Publication Date:  2006 Apr 
Date Detail:
Created Date:  2006-04-05     Completed Date:  2006-07-17     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  100937360     Medline TA:  Chembiochem     Country:  Germany    
Other Details:
Languages:  eng     Pagination:  631-7     Citation Subset:  IM    
Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, British Columbia, V6T 1Z1, Canada.
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MeSH Terms
Binding Sites / physiology
Carbohydrate Conformation
Chondroitin Lyases / chemistry*,  drug effects,  genetics
Monosaccharides / chemical synthesis,  chemistry
Time Factors
Tyrosine / chemistry*,  genetics
Uronic Acids / chemical synthesis,  chemistry
Reg. No./Substance:
0/Monosaccharides; 0/Protons; 0/Uronic Acids; 0/phenyl-4-O-(2',4'-dinitrophenyl)-beta-D-glucopyranosiduronic acid; 55520-40-6/Tyrosine; EC 4.2.2.-/Chondroitin Lyases

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