tailieunhanh - Báo cáo khoa học: The phosphate site of trehalose phosphorylase from Schizophyllum communeprobed by site-directed mutagenesis and chemical rescue studies

Schizophyllum commune a,a-trehalose phosphorylase utilizes a glycosyl-transferase-like catalytic mechanism to convert its disaccharide substrate into a-d-glucose 1-phosphate and a-d-glucose. Recruitment of phosphate by the free enzyme inducesa,a-trehalose binding recognition and promotes the catalytic steps. | ỊFEBS Journal The phosphate site of trehalose phosphorylase from Schizophyllum commune probed by site-directed mutagenesis and chemical rescue studies Christiane Goedl and Bernd Nidetzky Institute of Biotechnology and BiochemicalEngineering Graz University of Technology Austria Keywords catalytic mechanism chemical rescue family GT-4 glycosyltransferase a-retaining glucosyltransfer Correspondence B. Nidetzky Institute of Biotechnology and Biochemical Engineering Graz University of Technology Petersgasse 12 1 8010 Graz Austria Fax 43 316 873 8434 Tel 43 316 873 8400 E-mail Received 5 October 2007 revised 10 December 2007 accepted 19 December 2007 doi Schizophyllum commune a a-trehalose phosphorylase utilizes a glycosyltransferase-like catalytic mechanism to convert its disaccharide substrate into a-D-glucose 1-phosphate and a-D-glucose. Recruitment of phosphate by the free enzyme induces a a-trehalose binding recognition and promotes the catalytic steps. Like the structurally related glycogen phosphorylase and other retaining glycosyltransferases of fold family GT-B the trehalose phosphorylase contains an Arg507-XXXX-Lys512 consensus motif where X is any amino acid comprising key residues of its putative phosphate-binding sub-site. Loss of wild-type catalytic efficiency for reaction with phosphate kcat Km 21 000 M-1-s-1 was dramatic 107-fold in purified Arg507 fi Ala R507A and Lys512 fi Ala K512A enzymes reflecting a corresponding change of comparable magnitude in kcat Arg507 and Km Lys512 . External amine and guanidine derivatives selectively enhanced the activity of the K512A mutant and the R507A mutant respectively. Analysis of the pH dependence of chemical rescue of the K512A mutant by propargylamine suggested that unprotonated amine in combination with H2PO4 . the protonic form of phosphate presumably utilized in enzymatic catalysis caused restoration of activity. Transition state-like inhibition of the