Reversible modification of the sulfhydryl groups of Escherichia coli succinic thiokinase with methanethiolating reagents, 5,5′-dithio-bis(2-nitrobenzoic acid), p-hydroxymercuribenzoate, and ethylmercurithiosalicylate

Jonathan S. Nishimura, George L. Kenyon, Daniel J. Smith

Research output: Contribution to journalArticlepeer-review

25 Scopus citations

Abstract

Inhibition of Escherichia coli succinic thiokinase by either methyl methanethiolsulfonate (MeS-SO2Me) or by methoxycarbonylmethyl disulfide was reversed completely by addition of tributylphosphine, but not by incubation with dithiothreitol. At a point where about 4 moles of MeS- were incorporated per mole of enzyme (Mr 140,000), no loss of antigenicity (as measured by microcomplement fixation) was observed, but at least 80% of thiokinase activity was lost. No significant conformational change was indicated for this methanethiolated enzyme in the ultracentrifuge. In contrast, losses of both thiokinase activity and antigenicity were brought about by incubation with 5,5′-dithiobis 2-nitrobenzoic acid) (Nbs2), ethylmercurithiosalicylate, and p-hydroxymercuribenzoate. These losses were restored totally by incubation of modified enzyme with dithiothreitol but not with tributylphosphine. Preincubation of succinic thiokinases with MeSSO2Me and with Nbs2 protected the enzyme partially, and to the same degree, against the inhibiting effects of N-ethylmaleimide. These data suggest that, while both MeSSO2Me and Nbs2 may attack identical thiol groups on the enzyme, each may also react specifically with other thiol groups. Inhibition by MeS-SO2Me and by Nbs2 both appear not to be accompanied by dissociation into subunits and, in the later case, significant aggregation appears to be involved.

Original languageEnglish (US)
Pages (from-to)461-467
Number of pages7
JournalArchives of Biochemistry and Biophysics
Volume170
Issue numberC
DOIs
StatePublished - 1975
Externally publishedYes

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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