Catalytic residues Lys197 and Arg199 of Bacillus subtilis phosphoribosyl diphosphate synthase. Alanine-scanning mutagenesis of the flexible catalytic loop.

TY - JOUR

T1 - Catalytic residues Lys197 and Arg199 of Bacillus subtilis phosphoribosyl diphosphate synthase. Alanine-scanning mutagenesis of the flexible catalytic loop.

AU - Hove-Jensen, Bjarne

AU - Bentsen, Ann-Kristin K

AU - Harlow, Kenneth W

N1 - Keywords: Alanine; Alleles; Arginine; Bacillus subtilis; Catalysis; Enzyme Inhibitors; Kinetics; Lysine; Mutagenesis; Pliability; Ribose-Phosphate Pyrophosphokinase

PY - 2005

Y1 - 2005

N2 - 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 V(max) 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 K(m) values for ATP and ribose 5-phosphate of the two mutant enzymes were essentially unchanged. V(app) values of the remaining mutant enzymes were much less affected, ranging from 20 to 100% of the V(max) value of the wildtype enzyme. The data presented show that Lys197 and Arg199 are important in stabilization of the transition state.

AB - 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 V(max) 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 K(m) values for ATP and ribose 5-phosphate of the two mutant enzymes were essentially unchanged. V(app) values of the remaining mutant enzymes were much less affected, ranging from 20 to 100% of the V(max) value of the wildtype enzyme. The data presented show that Lys197 and Arg199 are important in stabilization of the transition state.

U2 - 10.1111/j.1742-4658.2005.04785.x

DO - 10.1111/j.1742-4658.2005.04785.x

M3 - Journal article

C2 - 16008562

SN - 1742-464X

VL - 272

SP - 3631

EP - 3639

JO - F E B S Journal

JF - F E B S Journal

IS - 14

ER -