Biosynthesis of type 3 capsular polysaccharide in Streptococcus pneumoniae. Enzymatic chain release by an abortive translocation process

The type 3 polysaccharide synthase from Streptococcus pneumoniae catalyzes sugar transfer from UDP-Glc and UDP-glucuronic acid (GlcUA) to a polymer with the repeating disaccharide unit of [3)-beta-d-GlcUA-(1-->4)-beta-d-Glc-(1-->]. Evidence is presented that release of the polysaccharide chains from S. pneumoniae membranes is time-, temperature-, and pH-dependent and saturable with respect to specific catalytic metabolites. In these studies, the membrane-bound synthase was shown to catalyze a rapid release of enzyme-bound polysaccharide when either UDP-Glc or UDP-GlcUA alone was present in the reaction. Only a slow release of polysaccharide occurred when both UDP sugars were present or when both UDP sugars were absent. Chain size was not a specific determinant in polymer release. The release reaction was saturable with increasing concentrations of UDP-Glc or UDP-GlcUA, with respective apparent K(m) values of 880 and 0.004 micrometer. The apparent V(max) was 48-fold greater with UDP-Glc compared with UDP-GlcUA. The UDP-Glc-actuated reaction was inhibited by UDP-GlcUA with an approximate K(i) of 2 micrometer, and UDP-GlcUA-actuated release was inhibited by UDP-Glc with an approximate K(i) of 5 micrometer. In conjunction with kinetic data regarding the polymerization reaction, these data indicate that UDP-Glc and UDP-GlcUA bind to the same synthase sites in both the biosynthetic reaction and the chain release reaction and that polymer release is catalyzed when one binding site is filled and the concentration of the conjugate UDP-precursor is insufficient to fill the other binding site. The approximate energy of activation values of the biosynthetic and release reactions indicate that release of the polysaccharide occurs by an abortive translocation process. These results are the first to demonstrate a specific enzymatic mechanism for the termination and release of a polysaccharide.