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Does 2-hydroxy-5-nitrobenzyl bromide react with the epsilon-subunit of the mitochondrial F1-ATPase?
A Baracca1, D Menegatti, G Parenti Castelli
1Department of Biology, University of Bologna, Italy.
Abstract:
The incubation of bovine mitochondrial F1-ATPase with 2-hydroxy-5-nitrobenzyl bromide (HNB), a selective reagent toward tryptophan residues in proteins, produced a concentration dependent inactivation of the enzyme and the covalent binding of 0.88 mol reagent/mol F1. Although HNB is highly specific for tryptophan it has also some reactivity toward cysteine, then a pre-treatment of F1 with several sulphydryl reagents has been performed to make the site of reaction clearer. This pre-treatment had neither effects in the binding stoichiometry nor in the extent of catalytic inhibition, suggesting that readly accessible thiol groups are not involved in the reaction with HNB. Since the only tryptophan bearing polypeptide of the bovine mitochondrial F1-ATPase complex is its smallest subunit, subunit-epsilon, this is the most probable candidate for HNB reaction. Therefore it may be inferred that the intactness and/or the correct conformation of this subunit could be important factor(s) for the multisite ATP hydrolytic activity of the enzyme.
Insights
Bovine mitochondrial F1-ATPase inactivation by HNB suggests subunit-epsilon is crucial. Its intactness and conformation are vital for the enzyme's ATP hydrolytic activity.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Bovine mitochondrial F1-ATPase is a key enzyme in cellular energy production.
- Understanding the functional roles of its subunits is essential for elucidating enzyme mechanisms.
Purpose of the Study:
- To investigate the role of tryptophan residues in bovine mitochondrial F1-ATPase activity.
- To identify the specific subunit involved in inactivation by 2-hydroxy-5-nitrobenzyl bromide (HNB).
Main Methods:
- Enzyme inactivation assays using HNB.
- Chemical modification of cysteine residues prior to HNB treatment.
- Stoichiometric analysis of reagent binding.
Main Results:
- HNB caused concentration-dependent inactivation of F1-ATPase, with 0.88 mol HNB binding per mol F1.
- Pre-treatment with sulfhydryl reagents did not alter HNB binding or inactivation extent.
- The smallest subunit, epsilon, was identified as the likely site of HNB reaction.
Conclusions:
- The epsilon subunit of bovine mitochondrial F1-ATPase is likely involved in the enzyme's catalytic activity.
- The intactness and proper conformation of the epsilon subunit are critical for multisite ATP hydrolysis.