Analysis of competition for substrate sites in an allosteric enzyme with co-operative kinetics. Effects of dAMP and dUMP on donkey spleen deoxycytidylate aminohydrolase.
Hypotheses about the interactions of effectors with conformations of allosteric enzymes having co-operative kinetics can be tested simply and exactly without knowledge of the nature of the intersubunit co-operativity by using a linkage approach to the analysis of steady-state kinetics. Applying this approach to competition for substrate sites in the allosteric enzyme donkey spleen dCMP aminohydrolase, we show that the kinetics are consistent with the hypothesis that the substrate dCMP and the competitor dAMP, as well as the allosteric activator dCTP, bind exclusively to the same conformation of the enzyme subunits. The linkage test can be applied in the presence of other effectors without knowledge of how these interact with the enzyme. Our tests showed that dCMP and DAMP are still bound exclusively to this same conformation in the presence of the product dUMP or of the allosteric inhibitor dTTP. We give evidence that dUMP binds to the same conformation as dCMP, but that it is also bound to other conformation(s). The advantages of the linkage approach, and some general problems in steady-state kinetics of allosteric enzymes, are discussed.
D000495:Allosteric Site / Q000187:drug effects; D000818:Animals; D001667:Binding, Competitive; D003844:DCMP Deaminase / Q000378:metabolism*; D003838:Deoxyadenine Nucleotides / Q000494:pharmacology*; D003845:Deoxycytosine Nucleotides / Q000494:pharmacology; D003856:Deoxyuracil Nucleotides / Q000494:pharmacology*; D007700:Kinetics; D008954:Models, Biological; D009709:Nucleotide Deaminases / Q000378:metabolism*; D010527:Perissodactyla; D011487:Protein Conformation; D013154:Spleen / Q000201:enzymology*; D013379:Substrate Specificity; D013942:Thymine Nucleotides / Q000494:pharmacology
D003838:Deoxyadenine Nucleotides; D003845:Deoxycytosine Nucleotides; D003856:Deoxyuracil Nucleotides; D013942:Thymine Nucleotides; C012873:2'-deoxy-5'-adenosine monophosphate; D009709:Nucleotide Deaminases; D003844:DCMP Deaminase; C024157:thymidine 5'-triphosphate