Cross-Linking and N-(1-Pyrenyl)maleimide Labeling of Cysteine Mutants of Proton-Pumping Pyridine Nucleotide Transhydrogenase of Escherichia coli
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- 作者：Edward G. Sedgwick ; Johan Meuller ; Cynthia Hou ; Jan Rydströ ; m ; and Philip D. Bragg
- 刊名：Biochemistry
- 出版年：1997
- 出版时间：December 9, 1997
- 年：1997
- 卷：36
- 期：49
- 页码：15285 - 15293
- 全文大小：196K
- 年卷期：v.36,no.49(December 9, 1997)
- ISSN：1520-4995
文摘
The pyridine nucleotide transhydrogenase of Escherichiacoli is a proton pump composed oftwo subunits (
and 
) organized as an22 tetramer. The enzyme containsseven cysteine residues, fivein the -subunit and two in the -subunit. The reaction ofthese residues with the cross-linking agentcupric 1,10-phenanthrolinate and with the fluorescent thiolreagent N-(1-pyrenyl)maleimide wasinvestigatedin mutants in which one or more of these cysteine residues had beenmutated to serine or threonineresidues. Mutation of Cys395 and Cys397 prevented disulfidebond formation to give the cross-linked 2 dimer. We concluded that the two-subunits of the holoenzyme interface in the region ofthese two cysteine residues. Pyrenylmaleimide reacted withdetergent-washed cytoplasmic membranevesicles containing high levels of transhydrogenase protein to showcharacteristic fluorescence emissionbands at 378-379, 397-398, and 419-420 nm. At higher ratiosof pyrenylmaleimide:transhydrogenase(>5:1) and longer times of reaction, an eximer band at 470 nm wasformed. This was attributed tointeraction between noncovalently bound molecules of pyrenylmaleimide.The cysteine residues of the-subunit (Cys147 and Cys260) were covalently modified bypyrenylmaleimide. Cys147 reactedmore strongly than Cys260 with the fluorophore, and the pyrenederivative of Cys147 was moreaccessible to quenching by 5-doxylstearate, suggesting a proximity tothe surface of the membrane.Covalent modification of Cys260 resulted in inhibition ofenzyme activity. The inhibition was attributedto the introduction of the bulky pyrene group into theenzyme.
and ) organized as an22 tetramer. The enzyme containsseven cysteine residues, fivein the -subunit and two in the -subunit. The reaction ofthese residues with the cross-linking agentcupric 1,10-phenanthrolinate and with the fluorescent thiolreagent N-(1-pyrenyl)maleimide wasinvestigatedin mutants in which one or more of these cysteine residues had beenmutated to serine or threonineresidues. Mutation of Cys395 and Cys397 prevented disulfidebond formation to give the cross-linked 2 dimer. We concluded that the two-subunits of the holoenzyme interface in the region ofthese two cysteine residues. Pyrenylmaleimide reacted withdetergent-washed cytoplasmic membranevesicles containing high levels of transhydrogenase protein to showcharacteristic fluorescence emissionbands at 378-379, 397-398, and 419-420 nm. At higher ratiosof pyrenylmaleimide:transhydrogenase(>5:1) and longer times of reaction, an eximer band at 470 nm wasformed. This was attributed tointeraction between noncovalently bound molecules of pyrenylmaleimide.The cysteine residues of the-subunit (Cys147 and Cys260) were covalently modified bypyrenylmaleimide. Cys147 reactedmore strongly than Cys260 with the fluorophore, and the pyrenederivative of Cys147 was moreaccessible to quenching by 5-doxylstearate, suggesting a proximity tothe surface of the membrane.Covalent modification of Cys260 resulted in inhibition ofenzyme activity. The inhibition was attributedto the introduction of the bulky pyrene group into theenzyme.