Probing the allosteric activation of pyruvate carboxylase using 2′,3′-0-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA

被引:13
作者
Adina-Zada, Abdussalam [1 ]
Hazra, Rasmani [1 ]
Sereeruk, Chutima [2 ]
Jitrapakdee, Sarawut [2 ]
Zeczycki, Tonya N. [3 ]
St Maurice, Martin [4 ]
Cleland, W. Wallace [3 ]
Wallace, John C. [5 ]
Attwood, Paul V. [1 ]
机构
[1] Univ Western Australia, Sch Biomed Biomol & Chem Sci, Crawley, WA 6009, Australia
[2] Mahidol Univ, Fac Sci, Dept Biochem, Mol Metab Res Grp, Bangkok 10400, Thailand
[3] Univ Wisconsin, Dept Biochem, Madison, WI 53726 USA
[4] Marquette Univ, Dept Biol Sci, Milwaukee, WI 53201 USA
[5] Univ Adelaide, Sch Mol & Biomed Sci, Adelaide, SA 5005, Australia
基金
澳大利亚研究理事会;
关键词
Pyruvate carboxylase; Allosteric activation; 2 ',3 '-0-(2,4,6-Trinitrophenyl) adenosine 5 '-triphosphate; Fluorescence stopped-flow; HALF-SITES REACTIVITY; STEADY-STATE KINETICS; BIOTIN CARBOXYLASE; DILUTION INACTIVATION; CATALYTIC ACTIVITY; MECHANISM; ENZYME; ATP; INSIGHTS; PEPTIDE;
D O I
10.1016/j.abb.2011.03.006
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:117 / 126
页数:10
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