Dynamic reactivities of dextransucrase.

Area of Science:

• Enzymology
• Carbohydrate Chemistry
• Biochemistry

Background:

• Dextransucrase from Streptococcus sanguis ATCC 10558 is an enzyme that synthesizes dextran from sucrose.
• Previous studies utilized pulse-chase experiments to investigate enzyme-bound oligosaccharides.

Purpose of the Study:

• To investigate the unexpected reactivity of immobilized dextransucrase previously charged with labeled sucrose.
• To elucidate the mechanism of glucosyl residue transfer and oligosaccharide metabolism by dextransucrase.

Main Methods:

• Immobilization of dextransucrase on hydroxylapatite.
• Pulsing the immobilized enzyme with labeled sucrose.
• Pulse-chase experiments using low concentrations of unlabeled sucrose.
• Analysis of radioactive labeling at the reducing termini of enzyme-bound oligosaccharides.
• Enzymatic assays with various acceptors like maltose, water, and fructose.

Main Results:

• Contrary to previous findings, low concentrations of unlabeled sucrose in the chase led to an increase in radioactivity at the reducing termini.
• Dextransucrase was shown to depolymerize bound oligosaccharides.
• Individual glucosyl residues were transferred to acceptors (maltose, water, fructose) forming trisaccharides, glucose, and sucrose.
• Oligosaccharides were slowly released from the enzyme.
• The reverse reaction, sucrose synthesis from transferred glucose residues and fructose, was also observed.

Conclusions:

• Dextransucrase exhibits complex reactivity beyond dextran synthesis, including oligosaccharide depolymerization and glucosyl transfer.
• The enzyme can act as a glucosyltransferase, transferring residues to water (hydrolysis) or other carbohydrates.
• The enzyme also demonstrates the ability to synthesize sucrose from transferred glucose residues and fructose.