Mechanistic studies on glycine reductase from Clostridium sticklandii
Mechanistic studies on glycine reductase from Clostridium sticklandii
The partially purified glycine reductase complex of Clostridium sticklandii catalyses the reductive deamination of glycine to acetate with concomitant formation of ATP from ADP plus phosphate in a soluble system. The reducing equivalents required are derived in vivo from oxidative reactions of amino acids via membrane associated electron transport systems, but may be provided by dithiols in vitro. The observation of previous workers that arsenate can replace phosphate as a substrate as in other substrate level phosphorylation reactions was confirmed. In order to carry out experiments on the mechanism of glycine reductase it was necessary to devise a new purification procedure since the one currently available resulted in partial inactivation of the enzyme. The mechanism of phosphorylation of ADP was investigated using two approaches. It was demonstrated that acetyl phosphate is not an intermediate in the reaction of labelling the oxygen atoms of the substrate glycine with 18O and recovering an 18O acetate species indicating that no ester formation and hydrolysis had taken place. The second approach involved the use of 18O phosphate as a substrate for glycine reductase in order to demonstrate that the formation of ATP occurs by a nucleophilic attack by an oxygen atom of the γ-phosphate of ADP on the phosphorus atom of phosphate. The activity exhibited by glycine reductase using thiophosphate as a substrate instead of phosphate was measured and found to be too low to allow the use of such phosphate analogues in stereochemical studies. The results obtained are consistent with the mechanism proposed by Barnard and Akhtar, (1979), which involves the formation of a lq`high energy' phosphorylated intermediate, with phosphate bound to a prosthetic group of the enzyme. (D68634/86)
University of Southampton
1985
Newport, Peter James
(1985)
Mechanistic studies on glycine reductase from Clostridium sticklandii.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
The partially purified glycine reductase complex of Clostridium sticklandii catalyses the reductive deamination of glycine to acetate with concomitant formation of ATP from ADP plus phosphate in a soluble system. The reducing equivalents required are derived in vivo from oxidative reactions of amino acids via membrane associated electron transport systems, but may be provided by dithiols in vitro. The observation of previous workers that arsenate can replace phosphate as a substrate as in other substrate level phosphorylation reactions was confirmed. In order to carry out experiments on the mechanism of glycine reductase it was necessary to devise a new purification procedure since the one currently available resulted in partial inactivation of the enzyme. The mechanism of phosphorylation of ADP was investigated using two approaches. It was demonstrated that acetyl phosphate is not an intermediate in the reaction of labelling the oxygen atoms of the substrate glycine with 18O and recovering an 18O acetate species indicating that no ester formation and hydrolysis had taken place. The second approach involved the use of 18O phosphate as a substrate for glycine reductase in order to demonstrate that the formation of ATP occurs by a nucleophilic attack by an oxygen atom of the γ-phosphate of ADP on the phosphorus atom of phosphate. The activity exhibited by glycine reductase using thiophosphate as a substrate instead of phosphate was measured and found to be too low to allow the use of such phosphate analogues in stereochemical studies. The results obtained are consistent with the mechanism proposed by Barnard and Akhtar, (1979), which involves the formation of a lq`high energy' phosphorylated intermediate, with phosphate bound to a prosthetic group of the enzyme. (D68634/86)
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Published date: 1985
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Local EPrints ID: 461711
URI: http://eprints.soton.ac.uk/id/eprint/461711
PURE UUID: e98d89fc-6aa7-40b9-b299-4c924b0ee186
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Date deposited: 04 Jul 2022 18:52
Last modified: 04 Jul 2022 18:52
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Author:
Peter James Newport
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