Covalent modification of enzymes by chloromethylketone derivatives of fatty acids
Covalent modification of enzymes by chloromethylketone derivatives of fatty acids
The work developed the synthesis of chloromethylketone fatty acid derivatives (Cl.CH2.CO.(CH2)n 000H]. Synthetic methods were developed for synthesis of these compounds with 2-8 methylene groups. Additional synthetic reactions are described for the synthesis of non-hydrolysable CoA esters, spin-label derivatives and iodo-ketones. Initially the reaction of S-chloro-4-oxopentanoic acid with rabbit muscle pyruvate kinase was studied. Pyruvate kinase was irreversibly inactivated with a pKa of 9.2. Inhibition was time and concentration dependent (Kr, 0.54 min-l; KI, 9.3 mM; n, 1.5 ± 0.2). Active site ligands prevented inhibition and their effectiveness was in the order Mg2+> phosphoenolpyruvate >ATP >> ADP > pyruvate. 5-Chloro-4-oxo-[3,5-'H] pentanoic acid was covalently bound to pyruvate kinase and demonstrated a stoichiometry of 1 mol of inhibitor bound per mol of pyruvate kinase subunit. The incorporation of inhibitor and the loss of enzyme activity was proportional. 4-Hydroxypentanoic acid alanine thioether was synthesised and identified as the modified amino acid formed by reacting pyruvate kinase with S-chloro4-oxo-(3,S-3H1 pentanoic acid. Performic acid oxidation of the labelled enzyme gave [(H] succinate (67%) and [3H] carboxymethylcysteine (33%) as predicted for the products of the Baeyer-Villiger reaction. NaBH4 reduction followed by periodate oxidation and analysis of radioactive formaldehyde production provided a convenient method for distinguishing between thiol and amino alkylation by halogenomethyl ketones. Chloromethylketone fatty acids were covalent inhibitors of pig heart acetoacetyl-CoA thiolase. The KI decreased by approximately 20 fold for each pair of methylenes added to the inhibitor chain length showing that the inhibitor initially bound to a non-polar region of the protein. This region may be at the active site since acetyl CoA and acetoacetyl CoA protected against inhibition. The inhibitor modified a thiol group on the enzyme since inactivation of the enzyme was prevented by reversible thiolmethylation of the active site thiol. Evidence was obtained that the enzyme active site thiol was particularly active.
University of Southampton
1981
Chalkley, Raymond Alan
(1981)
Covalent modification of enzymes by chloromethylketone derivatives of fatty acids.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
The work developed the synthesis of chloromethylketone fatty acid derivatives (Cl.CH2.CO.(CH2)n 000H]. Synthetic methods were developed for synthesis of these compounds with 2-8 methylene groups. Additional synthetic reactions are described for the synthesis of non-hydrolysable CoA esters, spin-label derivatives and iodo-ketones. Initially the reaction of S-chloro-4-oxopentanoic acid with rabbit muscle pyruvate kinase was studied. Pyruvate kinase was irreversibly inactivated with a pKa of 9.2. Inhibition was time and concentration dependent (Kr, 0.54 min-l; KI, 9.3 mM; n, 1.5 ± 0.2). Active site ligands prevented inhibition and their effectiveness was in the order Mg2+> phosphoenolpyruvate >ATP >> ADP > pyruvate. 5-Chloro-4-oxo-[3,5-'H] pentanoic acid was covalently bound to pyruvate kinase and demonstrated a stoichiometry of 1 mol of inhibitor bound per mol of pyruvate kinase subunit. The incorporation of inhibitor and the loss of enzyme activity was proportional. 4-Hydroxypentanoic acid alanine thioether was synthesised and identified as the modified amino acid formed by reacting pyruvate kinase with S-chloro4-oxo-(3,S-3H1 pentanoic acid. Performic acid oxidation of the labelled enzyme gave [(H] succinate (67%) and [3H] carboxymethylcysteine (33%) as predicted for the products of the Baeyer-Villiger reaction. NaBH4 reduction followed by periodate oxidation and analysis of radioactive formaldehyde production provided a convenient method for distinguishing between thiol and amino alkylation by halogenomethyl ketones. Chloromethylketone fatty acids were covalent inhibitors of pig heart acetoacetyl-CoA thiolase. The KI decreased by approximately 20 fold for each pair of methylenes added to the inhibitor chain length showing that the inhibitor initially bound to a non-polar region of the protein. This region may be at the active site since acetyl CoA and acetoacetyl CoA protected against inhibition. The inhibitor modified a thiol group on the enzyme since inactivation of the enzyme was prevented by reversible thiolmethylation of the active site thiol. Evidence was obtained that the enzyme active site thiol was particularly active.
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Published date: 1981
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Local EPrints ID: 459591
URI: http://eprints.soton.ac.uk/id/eprint/459591
PURE UUID: f5c4a56d-d116-4e47-a653-a4bce27a944f
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Date deposited: 04 Jul 2022 17:14
Last modified: 04 Jul 2022 17:14
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Author:
Raymond Alan Chalkley
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