Sucrose 6-alpha-D-glucosyltransferase from Streptococcus sobrinus: characterization of a glucosyl-enzyme complex

Journal Article · Tue Jan 24 04:00:00 EST 1989 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00428a006· OSTI ID:6075751

Mooser, G; Iwaoka, K R

A covalent glucosyl-enzyme was isolated from a quenched reaction of Streptococcus sobrinus sucrose 6-alpha-D-glucosyltransferase and radiolabeled sucrose. No complex was observed with heat-inactivated enzyme or when sucrose was replaced with radiolabeled maltose or glucose. The complex was stable at pH 2 in 1% sodium dodecyl sulfate, 6.0 M urea, and 4.0 M guanidine hydrochloride, but became increasingly labile with increased pH (32-min half-life at pH 7.0). D-Glucose was the exclusive radiolabeled compound identified when all radioactivity was released under mild alkaline conditions. Glucosyl-enzyme hydrolysis rates were linearly dependent on hydroxide ion concentration, giving a second-order rate constant of 2.15 x 10(5) M-1 min-1. When compared to the base lability of known glycosyl amino acid derivatives, the pH dependency of the glucosyl-enzyme most closely paralleled a glucosyl linkage to a carboxyl group. A novel application of a carbohydrate high-performance liquid chromatography column in aqueous solution was used to identify the anomeric form of D-glucose released on (i) alkaline hydrolysis of denatured glucosyl-enzyme and (ii) native enzyme hydrolysis of sucrose. The beta-anomer was identified in the former case and the alpha-anomer in the latter. The results with the denatured glucosyl-enzyme are consistent with a beta-glucosyl ester linkage to an aspartic or glutamic acid that hydrolyzes at the ester carbon with retention of anomeric configuration; for native glucosyltransferase catalysis, the data are consistent with a beta-glucosyl covalent intermediate as well, where deglucosylation occurs by attack at the acetal carbon with anomeric inversion.

• Research Organization: Univ. of Southern California, Los Angeles (USA)
• OSTI ID: 6075751
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 28:2; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ALDEHYDES
BACTERIA
BIOLOGICAL HALF-LIFE
CARBOHYDRATES
CARBON ISOTOPES
CHEMICAL COMPOSITION
CHEMICAL REACTIONS
CHROMATOGRAPHY
DECOMPOSITION
DISACCHARIDES
ENZYMATIC HYDROLYSIS
ENZYMES
GLUCOSE
HEXOSES
HYDROLYSIS
ISOTOPE APPLICATIONS
ISOTOPE DILUTION
ISOTOPES
LIQUID COLUMN CHROMATOGRAPHY
LYSIS
MICROORGANISMS
MONOSACCHARIDES
OLIGOSACCHARIDES
ORGANIC COMPOUNDS
SACCHARIDES
SACCHAROSE
SEPARATION PROCESSES
SOLVOLYSIS
STREPTOCOCCUS
TRACER TECHNIQUES
TRANSFERASES