The purpose of this project is to investigate the molecular basis for substrate recognition by (beta1->4) galactosyltransferase - a member of a large group of related enzymes in which structure-function relationships are still poorly understood. The general approach to this problem will be to use recombant DNA methods to produce mutated forms of the enzyme and then to compare the substrate specificities of the mutant enzymes with that of the wild type of enzyme. Fluorescent energy transfer, difference spectra, circular dichroism and 19F-NMR methods will be used to detect substrate-induced conformation changes in the normal and mutated forms of the enzyme. The substrate binding sites of natural and mutated enzymes will be mapped with analogues of UDP-galactose and N-acetylglucosamine to compare their substrate specificities. The experimental data will be analyzed to determine whether the mutations have a direct effect on substrate binding by specific amino acid residues, or they cause changes in enzyme conformation. Also in this investigation, a systematic series of experiments will be performed to find the conditions required to crystallize a recombinant galactosyltransferase that is free of the microheterogeneity found in the naturally occurring enzyme. The crystallization experiments will be done in the presence and absence of substrates in order to further our future goal of using x-ray diffraction data to study the effects of substrate binding on the enzyme's tertiary structure. This investigation is designed to be a model for future related studies of substrate induced conformational changes occurring in other glycosyltransferases. Information about the molecular basis of glycosyltransferase-substrate recognition is not yet available but is required to understand how naturally occurring mutations, that appear to occur in this group of enzymes, can affect oligosaccharide production which in turn influences normal and abnormal cell functions. The information gained from these studies could be of practical value in reengineering the galactosyltransferase molecule to have new substrate specificities so that it can more efficiently perform glycosylation reactions that do not occur under natural conditions and, hence aid in the biosynthesis of complex carbohydrates which cannot currently be produced by conventional methods of organic synthesis.