Catalytic residues Lys197 and Arg199 of Bacillus 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 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.
Hove-Jensen
Bjarne
B
Department of Biological Chemistry, Institute of Molecular Biology and Physiology, University of Copenhagen, Denmark. hove@mermaid.molbio.ku.dk
Bentsen
Ann-Kristin K
AK
Harlow
Kenneth W
KW
D000409:Alanine / Q000235:genetics* / Q000378:metabolism; D000483:Alleles; D001120:Arginine / Q000235:genetics / Q000378:metabolism*; D001412:Bacillus subtilis / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D002384:Catalysis; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D008239:Lysine / Q000235:genetics / Q000378:metabolism*; D016296:Mutagenesis / Q000235:genetics*; D018583:Pliability; D012268:Ribose-Phosphate Pyrophosphokinase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D004791:Enzyme Inhibitors; D001120:Arginine; D012268:Ribose-Phosphate Pyrophosphokinase; D008239:Lysine; D000409:Alanine