Effect of Glutaredoxin and Protein Disulfide Isomerase on the Glutathione-Dependent Folding of Ribonuclease A

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• 作者：Margherita Ruoppolo ; Johanna Lundströ ; m-Ljung ; Fabio Talamo ; Piero Pucci ; and Gennaro Marino
• 刊名：Biochemistry
• 出版年：1997
• 出版时间：October 7, 1997
• 年：1997
• 卷：36
• 期：40
• 页码：12259 - 12267
• 全文大小：210K
• 年卷期：v.36,no.40(October 7, 1997)
• ISSN：1520-4995

文摘

Protein folding, associated with oxidation and isomerization ofdisulfide bonds, was studiedusing reduced and denatured RNase A (rd-RNase A) and mixed disulfidebetween glutathione and reducedRNase A derivative (GS-RNase A) as starting materials. Folding wasinitiated by addition of freeglutathione (GSH + GSSG) and was monitored by electrospray massspectrometry (ESMS) time-courseanalysis and recovery of the native catalytic activity. The ESMSanalysis permitted both the identificationand quantitation of the population of intermediates present during therefolding process. Refolding ofrd-RNase A and GS-RNase A was also performed in the presence ofglutaredoxin (Grx) and/or proteindisulfide isomerase (PDI). All the analyses indicate a pathway ofsequential reactions in the formationof native RNase A. First, the reduced protein reacts with a singleglutathione molecule to form a mixeddisulfide which then evolves to an intramolecular S-S bond viathiol-disulfide exchange. Only at thisstage, the intermediate containing one intramolecular S-S reacts witha further glutathione molecule,reiterating the process. An analogous mechanism occurs in therefolding of GS-RNase A. The structuralanalysis of the intermediates formed during the refolding of RNase Ashowed for the first time that Grxis actually able to catalyze both formation and reduction of mixeddisulfides involving glutatione. Inboth refolding processes, starting from either rd-RNase A or GS-RNaseA, Grx displays a significantcatalysis at the early stages of the process. Addition of PDI ledto a net catalysis of the entire processwithout appearing to alter the refolding pathway. In the presenceof both Grx and PDI, the two enzymesshowed a synergistic activity either starting from rd-RNase A, aspreviously reported [Lundström, J., andHolmgren, A. (1995) J. Biol. Chem. 270,7822-7828], or starting from GS-RNase A. Present datasuggestthat the synergistic effect can be explained assuming that Grx actuallyfacilitates PDI action by catalyzingformation or reduction of mixed disulfides. The mixed disulfidesare then rapidly converted intointramolecular disulfides in the presence of PDI. These steps arerepeated sequentially throughout thewhole refolding, resulting in an immediate formation of fully oxidizedspecies even at the very beginningof the reaction. Finally, a Grx mutant, C14S Grx, in which one ofthe active site cysteine residues (Cys14)had been replaced by serine, had a similar effect on the distributionof folding intermediates, comparedto the wild-type protein, thus demonstrating that Grx acts by amonothiol mechanism either in the reductionor in the oxidation step.