HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 277, Issue 34, 31179-31186, August 23, 2002

This Article Full Text Full Text (PDF) All Versions of this Article: 277/34/31179    most recent M201552200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Pojasek, K. Articles by Sasisekharan, R. Search for Related Content PubMed PubMed Citation Articles by Pojasek, K. Articles by Sasisekharan, R. Social Bookmarking                      What's this?

Biochemical Characterization of the Chondroitinase B Active Site^*

Kevin Pojasek^§, Rahul Raman^¶, Patrick Kiley, Ganesh Venkataraman, and Ram Sasisekharan^**

From the  Division of Bioengineering and Environmental Health and the  Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

Chondroitinase B from Flavobacterium heparinum is the only known lyase that cleaves the glycosaminoglycan, dermatan sulfate (DS), as its sole substrate. A recent co-crystal structure of chondroitinase B with a disaccharide product of DS depolymerization has provided some insight into the location of the active site and suggested potential roles of some active site residues in substrate binding and catalysis. However, this co-crystal structure was not representative of the actual enzyme-substrate complex, because the disaccharide product did not have the right length or the chemical structure of the minimal substrate (tetrasaccharide) involved in catalysis. Therefore, only a limited picture of the functional role of active site residues in DS depolymerization was presented in previous structural studies. In this study, by docking a DS tetrasaccharide into the proposed active site of the enzyme, we have identified novel roles of specific active site amino acids in the catalytic function of chondroitinase B. Our conformational analysis also revealed a unique, symmetrical arrangement of active site amino acids that may impinge on the catalytic mechanism of action of chondroitinase B. The catalytic residues Lys-250, Arg-271, His-272, and Glu-333 along with the substrate binding residues Arg-363 and Arg-364 were mutated using site-directed mutagenesis, and the kinetics and product profile of each mutant were compared with recombinant chondroitinase B. Mutating Lys-250 to alanine resulted in inactivation of the enzyme, potentially attributable to the role of the residue in stabilizing the carbanion intermediate formed during enzymatic catalysis. The His-272 and Glu-333 mutants showed diminished enzymatic activity that could be indicative of a possible role for one or both residues in the abstraction of the C-5 proton from the galactosamine. In addition, the Arg-364 mutant had an altered product profile after exhaustive digestion of DS, suggesting a role for this residue in defining the substrate specificity of chondroitinase B.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				V. Prabhakar, I. Capila, V. Soundararajan, R. Raman, and R. Sasisekharan Recombinant Expression, Purification, and Biochemical Characterization of Chondroitinase ABC II from Proteus vulgaris J. Biol. Chem., January 9, 2009; 284(2): 974 - 982. [Abstract] [Full Text] [PDF]

				G. Michel, K. Pojasek, Y. Li, T. Sulea, R. J. Linhardt, R. Raman, V. Prabhakar, R. Sasisekharan, and M. Cygler The Structure of Chondroitin B Lyase Complexed with Glycosaminoglycan Oligosaccharides Unravels a Calcium-dependent Catalytic Machinery J. Biol. Chem., July 30, 2004; 279(31): 32882 - 32896. [Abstract] [Full Text] [PDF]