Kinetics of nucleotide binding to pyruvate carboxylase.
The kinetics of nucleotide binding to pyruvate carboxylase have been studied by measuring the fluorescence changes that occur on the binding and release of FTP and FDP, which are fluorescent formycin analogues of ATP and ADP. The rate constants and equilibrium binding constants for both MgFTP and MgFDP binding to pyruvate carboxylase have been determined. From the kinetics of displacement of MgFTP by MgATP and binding of MgFTP in the presence of MgATP, the rate constants of MgATP binding were estimated. A slow component to the fluorescence changes was seen to occur after the initial rapid, bimolecular binding step, when formycin nucleotides were mixed with the enzyme. HCO3- and pyruvate were shown to quench the fluorescence of enzyme-bound MgFTP, but did not affect the affinity of the enzyme for the nucleotide. Acetyl CoA reduced the affinity of the enzyme for both MgFDP and MgFTP by about 3-fold by decreasing the association rate constants (by 25%) and increasing the dissociation rate constants (by 2-fold). In the absence of Mg2+ a very rapid component to FTP binding was observed that was complete within about 3 ms, but no fast component was observed comparable to that seen in the presence of 4.5 mM MgCl2. Increasing the [Mg2+] gradually abolished this very fast component of the binding, while the amplitude of the fast component increased, although the rate constant for this component did not appear to be strongly dependent on [Mg2+]. The rate constants of the slow component of Mg.formycin nucleotide binding did not appear to be dependent on nucleotide concentration.(ABSTRACT TRUNCATED AT 250 WORDS)
Geeves
M A
MA
Department of Biochemistry, University of Western Australia, Nedlands.
Branson
J P
JP
Attwood
P V
PV
D000105:Acetyl Coenzyme A / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001639:Bicarbonates / Q000494:pharmacology; D001665:Binding Sites; D001667:Binding, Competitive; D002645:Chickens; D005456:Fluorescent Dyes / Q000378:metabolism; D005573:Formycins / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008274:Magnesium / Q000378:metabolism; D008954:Models, Biological; D011766:Pyruvate Carboxylase / Q000378:metabolism*; D011773:Pyruvates / Q000494:pharmacology; D019289:Pyruvic Acid; D012265:Ribonucleotides / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D001639:Bicarbonates; D005456:Fluorescent Dyes; D005573:Formycins; D011773:Pyruvates; D012265:Ribonucleotides; C032552:formycin triphosphate; C020332:formycin diphosphate; D000105:Acetyl Coenzyme A; D019289:Pyruvic Acid; D000255:Adenosine Triphosphate; D011766:Pyruvate Carboxylase; D008274:Magnesium