tailieunhanh - Báo cáo khoa học: Probing the active site of Corynebacterium callunae starch phosphorylase through the characterization of wild-type and His334fiGly mutant enzymes

His334 facilitates catalysis byCorynebacterium callunaestarch phosphory-lase through selective stabilization of the transition state of the reaction, partly derived from a hydrogen bond between its side chain and the C-6 hydroxy group of the glucosyl residue undergoing transfer to and from phosphate. | ỊFEBS Journal Probing the active site of Corynebacterium callunae starch phosphorylase through the characterization of wild-type and His334fiGly mutant enzymes Alexandra Schwarz1 Lothar Brecker2 and Bernd Nidetzky1 1 Institute of Biotechnology and BiochemicalEngineering Graz University of Technology Austria 2 Institute of Organic Chemistry University of Vienna Austria Keywords a-retaining glucosyltransfer phosphorus NMR pyridoxal5 -phosphate saturation transfer difference NMR starch phosphorylase Correspondence B. Nidetzky Institute of Biotechnology and Biochemical Engineering Graz University of Technology Petersgasse 12 A-8010 Graz Austria Fax 43 316 873 8434 Tel 43 316 873 8400 E-mail Received 15 May 2007 revised 1 August 2007 accepted 6 August 2007 doi His334 facilitates catalysis by Corynebacterium callunae starch phosphorylase through selective stabilization of the transition state of the reaction partly derived from a hydrogen bond between its side chain and the C-6 hydroxy group of the glucosyl residue undergoing transfer to and from phosphate. We have substituted His334 by a Gly and measured the disruptive effects of the site-directed replacement on active site function using steady-state kinetics and NMR spectroscopic characterization of the cofactor pyridoxal 5 -phosphate and binding of carbohydrate ligands. Purified H334G showed and of wild-type catalytic center activity for phosphorolysis of maltopentaose kcatP s-1 and substrate binding affinity in the ternary complex with enzyme bound to phosphate Km 280 mm respectively. The 31P chemical shift of pyridoxal 5 -phosphate in the wild-type was pH-dependent and not perturbed by binding of arsenate. At pH it was not sensitive to the replacement His334 fi Gly. Analysis of interactions of a-D-glucose 1-phosphate and a-D-xylose 1-phosphate upon binding to wild-type and H334G phosphorylase derived from saturation transfer difference .