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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.
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页码:117 / 126
页数:10
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