Abstract
Glutaredoxin (Grx) contains a redox-active disulfide and catalyzes thiol-disulfide interchange reactions with specificity for GSH. The dithiol form of Grx reduces mixed disulfides involving GSH or protein disulfides. During oxidative refolding of 8 microM reduced and denatured ribonuclease RNase-(SH)8 in a redox buffer of 1 mM GSH and 0.2 mM GSSG to yield native RNase-(S2)4, a large number of GSH-mixed disulfide species are formed. A lag phase that precedes formation of folded active RNase at a steady-state rate was shortened or eliminated by the presence of a catalytic concentration (0.5 microM) of Escherichia coli Grx together with protein disulfide-isomerase (PDI), its procaryotic equivalent E. coli DsbA, or the PDI analogue the E. coli thioredoxin mutant protein P34H. A mutant Grx in which one of the active site cysteine residues (Cys-11 and Cys-14) had been replaced by serine, C14S Grx, had similar effect compared with its wild-type counterpart. This demonstrated that Grx acted by a monothiol mechanism involving only Cys-11 and that RNase-S-SG-mixed disulfides were the substrates. Grx displayed synergistic activity together with PDI only in GSH/GSSG redox buffers with sufficiently low redox potential (E'0 of -208 or -181 mV) to allow reduction of the active site of Grx. In refolding systems that do not depend on glutathione, like cystamine/cysteamine or in the presence of selenite (SeO3(2-)), no synergistic activity of Grx was observed with PDI. We conclude that Grx acts by reducing mixed disulfides between GSH and RNase that are rate-limiting in enzyme-catalyzed refolding.
Highlights
From the Medical Nobel Institute for Biochemistry, Medical Biochemistry and Biophysics Karolinska Institute, S-171 77 Stockholm, Sweden
Enzymatic catalysis of native disulfide bond formation in fully reduced, inactive RNase-(SH)8 was followed as the rate of hydrolysis of 2',3'-cCMP catalyzed by the product, which is native RNase-(S2)4' This method is useful to study early events in disulfide formation and gives a fast measure of the rate order
Synergy Effect between Glutaredoxin and protein disulfide-isomerase (PDI) in GSH-dependent Refolding of RNase-(SH)8-In the presence of 1 /LM PDI and an optimized redox buffer consisting of 1 mM GSH and 0.2 mM GSSG, a lag phase of about 7 min preceded any detectable RNase activity (Fig. lA)
Summary
Vol 270, No 14, Issue of April 7, pp, 7822-7828, 1995 Printed in U.S.A. Glutaredoxin Accelerates Glutathione-dependent Folding of Reduced Ribonuclease A Together with Protein Disulfide-isomerase*. During oxidative refolding of 8 ILM reduced and denatured ribonuclease RNase-(SH)s in a redox buffer of 1 roM GSH and 0.2 DIM GSSG to yield native RNase-(S2)4' a large number of GSH-mixed disulfide species are formed. Of Medical Biochemistry and Biophysics, Karolinska lnst., S-171 77 Stockholm, Sweden. Together with thioredoxin and protein disulfide-isomerase (PDI), Grx is a member of a growing superfamily of well characterized proteins that share a common fold and an exposed active site dithiolldisulfide This consists of a 14-member disulfide ring in the oxidized form that is located at the end of a f3-strand and followed by an a-helix. The mutant protein C14S Grx (Bushweller et al, 1992) was used to generate a complex with GSH as a mixed disulfide (Grx'SSG), the solution structure of
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