Biochemical characterization of the chondroitinase ABC I active site.
cABC I (chondroitinase ABC I) from Proteus vulgaris is a GalAG (galactosaminoglycan) depolymerizing lyase that cleaves its substrates at the glycosidic bond via beta-elimination. cABC I cleaves a particularly broad range of GalAG substrates, including CS (chondroitin sulphate), DS (dermatan sulphate) and hyaluronic acid. We recently cloned and recombinantly expressed cABC I in Escherichia coli, and completed a preliminary biochemical characterization of the enzyme. In the present study, we have coupled site-directed mutagenesis of the recombinant cABC I with a structural model of the enzyme-substrate complex in order to investigate in detail the roles of active site amino acids in the catalytic action of the enzyme. The putative catalytic residues His-501, Tyr-508, Arg-560 and Glu-653 were probed systematically via mutagenesis. Assessment of these mutants in kinetic and end-point assays provided direct evidence on the catalytic roles of these active-site residues. The crystal structure of the native enzyme provided a framework for molecular docking of representative CS and DS substrates. This enabled us to construct recombinant enzyme-substrate structural complexes. These studies together provided structural insights into the effects of the mutations on the catalytic mechanism of cABC I and the differences in its processing of CS and DS substrates. All His-501 mutants were essentially inactive and thereby implicating this amino acid to play the critical role of proton abstraction during catalysis. The kinetic data for Glu-653 mutants indicated that it is involved in a hydrogen bonding network in the active site. The proximity of Tyr-508 to the glycosidic oxygen of the substrate at the site of cleavage suggested its potential role in protonating the leaving group. Arg-560 was proximal to the uronic acid C-5 proton, suggesting its possible role in the stabilization of the carbanion intermediate formed during catalysis.
Prabhakar
Vikas
V
Division of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Raman
Rahul
R
Capila
Ishan
I
Bosques
Carlos J
CJ
Pojasek
Kevin
K
Sasisekharan
Ram
R
D000818:Animals; D001665:Binding Sites; D002236:Carbohydrate Conformation; D002384:Catalysis; D019765:Chondroitin ABC Lyase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006025:Glycosaminoglycans / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D011487:Protein Conformation; D011514:Proteus vulgaris / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins; D012754:Sharks; D013379:Substrate Specificity; D013552:Swine
D006025:Glycosaminoglycans; D011994:Recombinant Proteins; D019765:Chondroitin ABC Lyase