Binding of the substrates and the allosteric inhibitor adenosine 5'-diphosphate to phosphoribosylpyrophosphate synthetase from Salmonella typhimurium.
The binding of the substrates, ATP and ribose-5-P, and the most effective inhibitor, ADP, to phosphoribosylpyrophosphate synthetase from Salmonella typhimurium was characterized using equilibrium dialysis of these compounds labeled with 32P. In the absence of ribose-5-P, ATP, ADP, and the ATP analogue alpha,beta-methylene ATP each bind cooperatively with half-saturation at 50 to 90 microM and Hill coefficients of 1.5 to 2. We propose that all three compounds bind at the same set of sites, which are presumably the active sites. When ribose-5-P was added, methylene ATP and ADP binding at these sites became tighter (Kd approximately 3 to 6 microM at 10 mM ribose-5-P) and lost its cooperativity. In the presence of ribose-5-P, ADP, but not methylene ATP, bound to a second site with half-saturation at approximately 150 microM and a Hill coefficient greater than 3. This result confirms the existence of an allosteric ADP site, which was previously postulated from kinetic studies (Switzer, R. L., and Sogin, D. C. (1973) J. Biol. Chem. 248, 1063-1073). Binding of ribose-5-P could not be detected in the absence of nucleotides, but it was readily measured in their presence. The apparent Kd of ribose-5-P varied from greater than 1 mM to approximately 5 microM as the concentration of either ADP or methylene ATP was increased from 0 to 2 mM. Inhibition of the enzyme by action of ADP at both active and allosteric sites could be observed kinetically.
D000244:Adenosine Diphosphate / Q000494:pharmacology*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000494:Allosteric Regulation; D000495:Allosteric Site; D001665:Binding Sites; D007700:Kinetics; D010770:Phosphotransferases / Q000378:metabolism*; D011485:Protein Binding; D012268:Ribose-Phosphate Pyrophosphokinase / Q000378:metabolism*; D012267:Ribosemonophosphates / Q000494:pharmacology; D012486:Salmonella typhimurium / Q000201:enzymology*
D012267:Ribosemonophosphates; C005147:5'-adenylyl (beta,gamma-methylene)diphosphonate; C031626:ribose-5-phosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D012268:Ribose-Phosphate Pyrophosphokinase; C002630:alpha,beta-methyleneadenosine 5'-triphosphate