tailieunhanh - Báo cáo khoa học: Tracking interactions that stabilize the dimer structure of starch phosphorylase from Corynebacterium callunae Roles of Arg234 and Arg242 revealed by sequence analysis and site-directed mutagenesis

Glycogen phosphorylases (GPs) constitute a family of widely spread catabolica1,4-glucosyltransferases that are active as dimers of two identical, pyridoxal 5¢-phosphate-containing subunits. In GP fromCorynebacterium callunae, physiological concentrations of phosphate are required to inhibit dissociation of protomers and cause a 100-fold increase in kinetic stability of the functional quarternary structure. | Eur. J. Biochem. 270 2126-2136 2003 FEBS 2003 doi Tracking interactions that stabilize the dimer structure of starch phosphorylase from Corynebacterium callunae Roles of Arg234 and Arg242 revealed by sequence analysis and site-directed mutagenesis Richard Griessler1 2 3 Alexandra Schwarz1 3 Jan Mucha2 and Bernd Nidetzky1 3 1 Institute of Food Technology and 2Centre of Applied Genetics University of Agricultural Sciences Vienna Austria 3Institute of Biotechnology Graz University of Technology Austria Glycogen phosphorylases GPs constitute a family of widely spread catabolic a1 4-glucosyltransferases that are active as dimers of two identical pyridoxal 5 -phosphate-containing subunits. In GP from Corynebacterium callunae physiological concentrations of phosphate are required to inhibit dissociation of protomers and cause a 100-fold increase in kinetic stability of the functional quarternary structure. To examine interactions involved in this large stabilization we have cloned and sequenced the coding gene and have expressed fully active C. callunae GP in Escherichia coli. By comparing multiple sequence alignment to structurefunction assignments for regulated and nonregulated GPs that are stable in the absence of phosphate we have scrutinized the primary structure of C. callunae enzyme for sequence changes possibly related to phosphate-dependent dimer stability. Location of Arg234 Arg236 and Arg242 within the predicted subunit-to-subunit contact region made these residues primary candidates for site-directed muta genesis. Individual Arg fi Ala mutants were purified and characterized using time-dependent denaturation assays in urea and at 45 C. R234A and R242A are enzymatically active dimers and in the absence of added phosphate they display a sixfold and fourfold greater kinetic stability of quarternary interactions than the wild-type respectively. The stabilization by 10 mM of phosphate was however up to 20-fold greater in the .