期刊
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
卷 509, 期 2, 页码 117-126出版社
ELSEVIER SCIENCE INC
DOI: 10.1016/j.abb.2011.03.006
关键词
Pyruvate carboxylase; Allosteric activation; 2 ',3 '-0-(2,4,6-Trinitrophenyl) adenosine 5 '-triphosphate; Fluorescence stopped-flow
资金
- National Institute of Health [GM070455]
- Australian Research Council [DP0988153]
- Thailand Research Fund
- Faculty of Science, Mahidol University
- NIH, National Institute of Diabetes and Digestive and Kidney Diseases [F32DK083898]
- Australian Research Council [DP0988153] Funding Source: Australian Research Council
2',3'-0-(2,4,6-Trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP) is a fluorescent analogue of ATP. MgTNP-ATP was found to be an allosteric activator of pyruvate carboxylase that exhibits competition with acetyl CoA in activating the enzyme. There is no evidence that MgTNP-ATP binds to the MgATP substrate binding site of the enzyme. At concentrations above saturating, MgATP activates bicarbonate-dependent ATP cleavage, but inhibits the overall reaction. The fluorescence of MgTNP-ATP increases by about 2.5-fold upon binding to the enzyme and decreases on addition of saturating acetyl CoA. However, not all the MgTNP-ATP is displaced by acetyl CoA, or with a combination of saturating concentrations of MgATP and acetyl CoA. The kinetics of the binding of MgTNP-ATP to pyruvate carboxylase have been measured and shown to be triphasic, with the two fastest phases having pseudo first-order rate constants that are dependent on the concentration of MgTNP-ATP. The kinetics of displacement from the enzyme by acetyl CoA have been measured and also shown to be triphasic. A model of the binding process is proposed that links the kinetics of MgTNP-ATP binding to the allosteric activation of the enzyme. (C) 2011 Elsevier Inc. All rights reserved.
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