tailieunhanh - Báo cáo khoa học: Catalytic residues Lys197 and Arg199 ofBacillus subtilis phosphoribosyl diphosphate synthase Alanine-scanning mutagenesis of the flexible catalytic loop

Eleven of the codons specifying the amino acids of the flexible catalytic loop [KRRPRPNVAEVM(197–208)] of Bacillus subtilis phosphoribosyl diphosphate synthase have been changed individually to specify alanine. The resulting variant enzyme forms, as well as the wildtype enzyme, were produced in anEscherichia colistrain lacking endogenous phosphoribosyl diphosphate synthase activity and purified to near homogeneity. The B. subtilisphosphoribosyl diphosphate synthase mutant variants K197A and R199A were studied in detail | ềFEBS Journal Catalytic residues Lys197 and Arg199 of Bacillus subtilis phosphoribosyl diphosphate synthase Alanine-scanning mutagenesis of the flexible catalytic loop Bjarne Hove-Jensen Ann-Kristin K. Bentsen and Kenneth W. Harlow Department of BiologicalChemistry Institute of Molecular Biology and Physiology University of Copenhagen Denmark Keywords flexible loop nucleotide metabolism PRPP Correspondence B. Hove-Jensen Department of Biological Chemistry Institute of Molecular Biology and Physiology University of Copenhagen DK-1307 Copenhagen K Denmark Fax 45 3532 2040 Tel 45 3532 2027 E-mail hove@ Website http Present address Novo Nordisk A S Drug Metabolism Novo Nordisk Park DK-2760 Malov Denmark Received 7 April2005 revised 12 May 2005 accepted 20 May 2005 Eleven of the codons specifying the amino acids of the flexible catalytic loop KRRPRPNVAEVM 197-208 of Bacillus subtilis phosphoribosyl diphosphate synthase have been changed individually to specify alanine. The resulting variant enzyme forms as well as the wildtype enzyme were produced in an Escherichia coli strain lacking endogenous phosphoribosyl diphosphate synthase activity and purified to near homogeneity. The B. subtilis phosphoribosyl diphosphate synthase mutant variants K197A and R199A were studied in detail. The physical properties of the two enzymes were similar to those of the wildtype enzyme. Kinetic characterization showed that the Vmax values of the K197A and R199A mutant enzymes were more than 30 000- and more than 24 000-fold reduced respectively compared to the wildtype enzyme. The Km values for ATP and ribose 5-phosphate of the two mutant enzymes were essentially unchanged. Vapp values of the remaining mutant enzymes were much less affected ranging from 20 to 100 of the Vmax value of the wildtype enzyme. The data presented show that Lys197 and Arg199 are important in stabilization of the transition state. doi The compound .