Mechansim and specificity of alpha-glucan phosphorylase

Glycogen phosphorylases occur ubiquituously in Nature and play a pivotal role in cellular carbohydrate metabolism by providing rapidly assimilated carbon through the phosphorolysis of a-glucan substrates stockpiled in the cytosol or taken up from the extracellular environment. Research on glycogen phosphorylases has been seminal for many fields of modern biochemistry, structural and cell biology and led to decipher the complex molecular mechanims by which the cell exercises precise and finely tuned control of glycogen phosphorylase activity in response to extracellular signals using covalent phosphorylation processes and allosteric effectors. Mechanistically, glycogen phosphorylases can be classified as glycosyl transferases that catalyse glucosyl transfer from and to phosphate with retention of configuration in an axial-to-axial reaction. The absolute dependence of enzyme activity on the cofactor pyridoxal 5`-phosphate is unique feature of glycogen phosphorylases. In spite of the plethora of detailed information at a near-atomic level on the catalytic mechanism and structure/function relationships of glycogen phosphorylase, a number of interesting questions remain to be answered. They relate to the stabilisation of the putative oxocarbenium-ion like reaction intermediate, the molecular basis of polysaccharide specificity, the kinetic basis of the synergistic interaction of substrates at the active site, and the contribution of intersubunit interactions for activity, specificity and stability. The present projects would like to address these questions which are considered to be of considerable significance in the broad and highly competitive field of enzymatic glucosyl transfer. It aims at combining the contributions from site-directed mutagenesis, steady-state and pre-steady-state kinetic studies of substrate and ligand binding as well as the enzymatic glucosyl transfer reaction, and high-resolution structure analysis. Non-regulated bacterial starch phosphorylase from Corynebacterium callunae has been chosen as a promising new model to study these enzymatic features of glycogen phosphorylase. Specific research tasks and key goals of the project will be: 1) determine the role of histidine 377 in substrate binding and stabilisation of the oxocarbenium ion-like intermediateransition state of the reaction catalysed by glycogen phosphorylase using binding studies and if possible, kinetic measurements, and structure studies of selected mutants 2) identify possible changes in reaction mechanism upon replacement of the histidine by aspartate or glutamate (covalent intermediate formation), and by alanine or glycine (change to inverting mechanism in the presence of external nucleophiles such as azide) 3) determine the role of interfacial arginines, especially those conserved in starch phosphorylase and rabbit muscle phosphorylase, for catalytic function, substrate specificity, stability and stabilisation of the bacterial enzyme by phosphate 4) determine the mechanism of substrate binding in fast kinetic studies and provide basis of substrate synergism in phosphorylase 5) provide the basis of polysaccharide specificity of starch phosphorylase and unravel fundamentals of the communication between the active site and the polysaccharide binding site in the bacterial phosphorylase

Simple and complex carbohydrates have been described as `the last frontier of molecular and cell biology`. The carbohydrates, or often `the sugars`, are biomolecules characterised by enormous structural complexity and functional diversity. To a wider public, they are known mainly because they provide a major caloric portion of the human diet and sometimes impart a sweet taste to the product. However, physiological roles in which carbohydrates act as a `cellular language` have been unveiled and rely on the huge coding potential of the individual monosaccharides that constitute the functional structure. Among the plethora of carbohydrate-active enzymes, those which can catalyse the formation of specific linkages between the monosaccharide building blocks to yield oligosaccharides are especially challenging. This group of enzymes, functionally classified as `the glycosyltransferases`, is large, and its members differ in respect to both amino acid sequence and three-dimensional structure, partly reflecting the complexity of the reaction products of their catalytic action. The now completed project significantly advances the understanding of the molecular mechanism of glycosyltransferases. The evidence, which was obtained through studies of a special group of glycosyltransferases, the so-called phosphorylases, is of a general relevance as it can be extrapolated on the basis of known evolutionary relationships among various families of glycosyltransferases. It provides a very useful and strengthened basis for further applications of phosphorylases in the synthesis of rare glycosides and the development of mechanism-based therapeutic inhibitors of glycosyltransferases in general. The project examined by using an assortment of modern biochemical techniques with a special focus on kinetic methods the role of selected amino acids in three types of microbial phosphorylases and determined detailed structure-function relationships.