Size and subdomain architecture of the glucan-binding domain of sucrose:3-alpha-D-glucosyltransferase from Streptococcus sobrinus.
Mild trypsin proteolysis of Streptococcus sobrinus sucrose:3-alpha-D-glucosyltransferase (GTF-I) reduced most of the enzyme to small products but left a few large fragments undigested. The digest had no glucosyl transfer activity, but several digestion products had an affinity for glucan equivalent to that of the native enzyme. The glucan-binding fragments ranged in size from 17 to 60 kilodaltons (kDa), with a particularly prominent 42-kDa fragment. The largest of these (60 kDa) appears to be the full extent of the domain since it increases in abundance when the enzyme is protected with glucan during proteolysis. The presence of smaller fragments with glucan-binding function and intact tertiary structure indicates that the full domain must be built on glucan-binding subdomains. Among the range of glucan-binding fragments, only the 42-kDa segment could be satisfactorily purified. It was subjected to N-terminal sequence analysis and, because of some ambiguity, was also subjected to chymotrypsin digestion and sequence of several chymotryptic peptides. The sequence data established that the 42-kDa domain fragment is initiated approximately two-thirds into the 170-kDa enzyme in a region previously identified as a segment of the gene that includes the glucan-binding domain (J. J. Ferretti, M. L. Gilpin, and R. R. B. Russell, J. Bacteriol. 169:4271-4278, 1987). The site is approximately 60 kDa from the C terminus and covers a region characterized by extensive amino acid sequence repeats. The data are discussed in the context of the size range of the glucan-binding fragments and subdomain architecture of the full glucan-binding domain.
Wong
C
C
Department of Basic Sciences, University of Southern California School of Dentistry, Los Angeles 90089-0641.
Hefta
S A
SA
Paxton
R J
RJ
Shively
J E
JE
Mooser
G
G
D000595:Amino Acid Sequence; D001665:Binding Sites; D005936:Glucans / Q000378:metabolism*; D005964:Glucosyltransferases; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D010447:Peptide Hydrolases; D013291:Streptococcus / Q000201:enzymology*
D005936:Glucans; D010446:Peptide Fragments; C036655:1,3-alpha-D-glucan synthase; D005964:Glucosyltransferases; D010447:Peptide Hydrolases