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Biochemistry. 2006 Aug 1;45(30):9138-44.

Chemical mechanism and specificity of the C5-mannuronan epimerase reaction.

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  • 1Department of Biochemistry, University of Missouri-Columbia, Columbia, Missouri 65211, USA.

Abstract

C5-mannuronan epimerase catalyzes the formation of alpha-L-guluronate residues from beta-D-mannuronate residues in the synthesis of the linear polysaccharide alginate. The reaction requires the abstraction of a proton from C5 of the residue undergoing epimerization followed by re-protonation on the opposite face. Rapid-mixing chemical quench experiments were conducted to determine the nature of the intermediate formed upon proton abstraction in the reaction catalyzed by the enzyme from Pseudomonas aeruginosa. Colorimetric and HPLC analysis of quenched samples indicated that shortened oligosaccharides containing an unsaturated sugar residue form as transient intermediates in the epimerization reaction. This suggests that the carbanion is stabilized by glycal formation, concomitant with cleavage of the glycosidic bond between the residue undergoing epimerization and the adjacent residue. The time dependence of glycal formation suggested that slow steps flank the chemical steps in the catalytic cycle. Solvent isotope effects on V and V/K were unity, consistent with a catalytic cycle in which chemistry is not rate-limiting. The specificity of the epimerase with regard to neighboring residues was examined, and it was determined that the enzyme showed no bias for mannuronate residues adjacent to guluronates versus those adjacent to mannuronates. Proton abstraction and sugar epimerization were irreversible. Existing guluronate residues already present in the polysaccharide were not converted to mannuronates, nor was incorporation of solvent deuterium into existing mannuronates observed.

[PubMed - indexed for MEDLINE]
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