Self-assembly and catalytic activity of the pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus

被引:38
作者
Domingo, GJ [1 ]
Chauhan, HJ [1 ]
Lessard, IAD [1 ]
Fuller, C [1 ]
Perham, RN [1 ]
机构
[1] Univ Cambridge, Dept Biochem, Cambridge Ctr Mol Recognit, Cambridge CB2 1GA, England
来源
EUROPEAN JOURNAL OF BIOCHEMISTRY | 1999年 / 266卷 / 03期
关键词
multienzyme complex; pyruvate dehydrogenase complex; dihydrolipoyl acetyltransferase; self-assembly; competitive binding;
D O I
10.1046/j.1432-1327.1999.00966.x
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
The pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus was reconstituted in vitro from recombinant proteins derived from genes over-expressed in Escherichia coli, Titrations of the icosahedral (60-mer) dihydrolipoyl acetyltransferase (E2) core component with the pyruvate decarboxylase (E1, alpha(2)beta(2)) and dihydrolipoyl dehydrogenase (E3, alpha(2)) peripheral components indicated a variable composition defined predominantly by tight and mutually exclusive binding of E1 and E3 with the peripheral subunit-binding domain of each E2 chain. However, both analysis of the polypeptide chain ratios in complexes generated from various mixtures of E1 and E3. and displacement of E1 or E3 from E1-E2 or E3-E2 subcomplexes by E3 or E1, respectively, showed that the multienzyme complex does not behave as a simple competitive binding system. This implies the existence of secondary interactions between the E1 and E3 subunits and E2 that only become apparent on assembly. Exact geometrical distribution of E1 and E3 is unlikely and the results are best explained by preferential arrangements of E1 and E3 on the surface of the E2 core, superimposed on their mutually exclusive binding to the peripheral subunit-binding domain of the E2 chain. Correlation of the subunit composition with the overall catalytic activity of the enzyme complex confirmed the lack of any requirement for precise stoichiometry or strict geometric arrangement of the three catalytic sites and emphasized the crucial importance of the flexibility associated with the lipoyl domains and intramolecular acetyl group transfer in the mechanism of active-site coupling.
引用
收藏
页码:1136 / 1146
页数:11
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