Structure-based mutagenic analysis of mechanism and substrate specificity in mammalian glycosyltransferases: porcine ST3Gal-I.

Sialyltransferases (STs) play essential roles in signaling and in the cellular recognition processes of mammalian cells by selectively installing cell-surface sialic acids in an appropriate manner both temporally and organ-specifically. The availability of the first three-dimensional structure of a mammalian (GT29) sialyltransferase has, for the first time, allowed quantitative structure/function analyses to be performed, thereby providing reliable insights into the roles of key active site amino acids. Kinetic analyses of mutants of ST3Gal-I, in conjunction with structural studies, have confirmed the mechanistic roles of His302 and His319 as general acid and base catalysts, respectively, and have quantitated other interactions with the cytosine monophosphate-N-acetyl ß-neuraminic acid donor substrate. The contributions of side chains that provide key interactions with the acceptor substrate, defining its specificity, have also been quantitated. Particularly important transition-state interactions of 2.5 and 2.7 kcal mol(-1) are found between the acceptor axial 4-hydroxyl and the conserved side chains of Gln108 and Tyr269, respectively. These results provide a basis for the engineering of mammalian STs to accommodate non-natural substrate analogs that should prove valuable as chemical biological probes of sialyltransferase function.