Binding of purine nucleotides to two regulatory sites results in synergistic feedback inhibition of glutamine 5-phosphoribosylpyrophosphate amidotransferase.
Glutamine 5-phosphoribosylpyrophosphate amidotransferase from Escherichia coli is subject to synergistic feedback regulation by adenine and guanine nucleotides. Inhibition assays and equilibrium binding measurements have established that synergistic inhibition by AMP and GMP results from synergistic binding to two sites/enzyme subunit in the homotetramer. Although each nucleotide can bind to both sites, analyses of the wild type and mutant enzymes indicate that binding of GMP to an A (allosteric) site and AMP to a proximal C (catalytic) site are necessary for synergistic inhibition. K326Q and P410W amino acid replacements result in decreased binding affinity for GMP and AMP and lead to corresponding reductions in feedback inhibition. The K326Q A site mutation results not only in decreased affinity of GMP for the mutant A site but also has an adverse effect on AMP affinity for the C site. Similarly, the P410W C site mutation has a detrimental effect on binding of AMP to the mutant C site and also on affinity of GMP to the A site. The fact that a mutation in one site affects binding of nucleotides to both sites provides further evidence for synergistic binding of nucleotides.
Zhou
G
G
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907.
Smith
J L
JL
Zalkin
H
H
D000249:Adenosine Monophosphate / Q000378:metabolism* / Q000494:pharmacology; D000494:Allosteric Regulation; D000495:Allosteric Site; D000582:Amidophosphoribosyltransferase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D005246:Feedback; D006157:Guanosine Monophosphate / Q000378:metabolism* / Q000494:pharmacology; D007700:Kinetics; D008239:Lysine; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism
D011994:Recombinant Proteins; D000249:Adenosine Monophosphate; D006157:Guanosine Monophosphate; D000582:Amidophosphoribosyltransferase; D008239:Lysine