Biochemical characterization of the chondroitinase ABC I active site

AUTOR(ES)

Prabhakar, Vikas

FONTE

Portland Press Ltd.

RESUMO

cABC I (chondroitinase ABC I) from Proteus vulgaris is a GalAG (galactosaminoglycan) depolymerizing lyase that cleaves its substrates at the glycosidic bond via β-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.

Documentos Relacionados

• 8-Oxoguanine rearranges the active site of human topoisomerase I
• Calcium diffusion enhanced after cleavage of negatively charged components of brain extracellular matrix by chondroitinase ABC
• THE BIOCHEMICAL FUNCTION OF BIOTIN, VI. CHEMICAL STRUCTURE OF THE CARBOXYLATED ACTIVE SITE OF PROPIONYL CARBOXYLASE*†
• Biochemical characterization of a variant human medium-chain acyl-CoA dehydrogenase with a disease-associated mutation localized in the active site.
• Mapping the active-site tyrosine of vaccinia virus DNA topoisomerase I.