The role of sulfhydryl groups in the regulation of spinach leaf sucrose phosphate synthase

Douglas C. Doehlert, Steven C. Huber

Research output: Contribution to journalArticlepeer-review

Abstract

The activation of spinach leaf sucrose phosphate synthase by glucose 6-phosphate (Glc6 P) was labile in the absence of dithiothreitol, whereas enzyme activity in the absence of activator remained stable. The loss of regulation by Glc6P proceeded more rapidly by treatment with 24 mM H2O2 and was reversible by 2.5 mM dithiothreitol. Titration of the enzyme with the sulfhydryl reagents N-ethylmaleimide or p-chloromercuribenzenesulfonic acid (0-50 μM) also resulted in elimination of Glc6P activation and most of the inorganic phosphate inhibition without eliminating catalytic activity. The results suggested that Glc6P and phosphate bind to a modifier site that is distinct from the catalytic site. The modifier site apparently contains essential and accessible sulfhydryl groups, while the catalytic site does not. Even though sulfhydryl reagents did not eliminate catalysis, changes in substrate affinities occurred. Treatment with 50 μM p-chloromercuribenzenesulfonic acid lowered the Km (fructose 6-phosphate) from 3.0 to 1.0 mM and increased the Km (uridine diphosphate glucose) from 3.0 to 8.9 mM without affecting the Vmax. Spinach leaf sucrose phosphate synthase extracted in the absence of reductant from leaves in the light (at 15:00 h) or in the dark (at 03:00 h) exhibited similar Glc6P activation, suggesting that the light/dark status of leaves does not affect the redox status of essential sulfhydryl groups of the modifier site.

Original languageEnglish (US)
Pages (from-to)267-273
Number of pages7
JournalBiochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
Volume830
Issue number3
DOIs
StatePublished - Aug 23 1985

Keywords

  • (Spinach)
  • Enzyme regulation
  • Sucrose phosphate synthase
  • Sulfhydryl group

ASJC Scopus subject areas

  • Biophysics
  • Structural Biology
  • Biochemistry
  • Molecular Biology

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