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Dimeric pig heart succinate-coenzyme A transferase uses only one subunit to support catalysis

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Lloyd, Adrian J. and Shoolingin-Jordan, P. M. (2001) Dimeric pig heart succinate-coenzyme A transferase uses only one subunit to support catalysis. Biochemistry, 40 (8). pp. 2455-2467. doi:10.1021/bi002169v ISSN 0006-2960.

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Official URL: https://doi.org/10.1021/bi002169v

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Abstract

Pig heart succinate-coenzyme A transferase (succinyl-coenzyme A: 3-oxoacid coenzyme A transferase; E. C. 2.8.3.5.), a dimeric enzyme purified by affinity chromatography on Procion Blue MX-2G Sepharose, reacts with acetoacetyl-coenzyme A to form a covalent enzyme-coenzyme A thiolester intermediate in which the active site glutamate (E344) of both subunits each forms thiolester links with coenzyme A. Reaction of this dimeric enzyme-coenzyme A species with sodium borohydride leads to inactivation of the enzyme and reduction of the thiolester on both subunits to the corresponding enzyme alcohol, as judged by electrospray mass spectrometry. Reaction of the dimeric enzyme-coenzyme A intermediate with either succinate or acetoacetate, however, results in only one-half of the coenzyme A being transferred to the acceptor carboxylate to form either succinyl-coenzyme A or acetoacetyl-coenzyme A. Reaction of this latter enzyme species with borohydride caused no loss of enzyme activity despite the reduction of the remaining half of the enzyme-coenzyme A thiolester to the enzyme alcohol. That this catalytic asymmetry existed between subunits within the same enzyme dimer was demonstrated by showing that the enzyme species, created by successive reaction with acetoacetyl-coenzyme A and succinate, bound to Blue MX-2G Sepharose through the remaining available active site and could be eluted as a single chromatographic species by succinyl-coenzyme A. It is concluded that while both of the subunits of the succinate-coenzyme A transferase dimer are able to form enzyme-coenzyme A thiolester intermediates, only one subunit is competent to transfer the coenzyme A moiety to a carboxylic acid acceptor to form the new acyl-coenzyme A product. The possible structural basis for this catalytic asymmetry and its mechanistic implications are discussed.

Item Type: Journal Article
Divisions: Faculty of Science, Engineering and Medicine > Science > Life Sciences (2010- )
Journal or Publication Title: Biochemistry
Publisher: American Chemical Society
ISSN: 0006-2960
Official Date: 2 February 2001
Dates:
DateEvent
2 February 2001Published
29 December 2000Accepted
14 September 2000Submitted
Volume: 40
Number: 8
Page Range: pp. 2455-2467
DOI: 10.1021/bi002169v
Status: Peer Reviewed
Publication Status: Published
Access rights to Published version: Restricted or Subscription Access

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