Comparison of the activities of protein disulphide-isomerase and thioredoxin in catalysing disulphide isomerization in a protein substrate

Hawkins, Hilary C.; Blackburn, E. K.; Freedman, Robert B.

doi:10.1042/bj2750349

Cited by 112 publications

(58 citation statements)

References 39 publications

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“…Correct protein folding is considered to be catalyzed by protein-disulfide isomerase (26), which is a strong oxidant. However, thioredoxin was also shown to possess a disulfide isomerase activity (27). The present work suggests that thioredoxin could also be involved in the isomerization of disulfides in conjunction with its disulfide reducing function.…”

Section: Table III Kinetic Parameters Of Mutant Nadp-mdhsmentioning

confidence: 85%

An Internal Cysteine Is Involved in the Thioredoxin-dependent Activation of Sorghum Leaf NADP-malate Dehydrogenase

Ruelland

Lemaire-Chamley

Maréchal

et al. 1997

Journal of Biological Chemistry

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The chloroplastic NADP-malate dehydrogenase is activated by thiol/disulfide interchange with reduced thioredoxins. Previous experiments showed that four cysteines located in specific N-and carboxyl-terminal extensions were implicated in this process, leading to a model where no internal cysteine was involved in activation. In the present study, the role of the conserved four internal cysteines was investigated. Surprisingly, the mutation of cysteine 207 into alanine yielded a protein with accelerated activation time course, whereas the mutations of the three other internal cysteines into alanines yielded proteins with unchanged activation kinetics. These results suggested that cysteine 207 might be linked in a disulfide bridge with one of the four external cysteines, most probably with one of the two amino-terminal cysteines whose mutation similarly accelerates the activation rate. To investigate this possibility, mutant malate dehydrogenases (MDHs) where a single amino-terminal cysteine was mutated in combination with the mutation of both carboxyl-terminal cysteines were produced and purified. The C29S/C365A/ C377A mutant MDH still needed activation by reduced thioredoxin, while the C24S/C365A/C377A mutant MDH exhibited a thioredoxin-insensitive spontaneous activity, leading to the hypothesis that a Cys 24 -Cys 207 disulfide bridge might be formed during the activation process. Indeed, an NADP-MDH where the cysteines 29, 207, 365, and 377 are mutated yielded a permanently active enzyme very similar to the previously created permanently active C24S/C29S/C365A/C377A mutant. A two-step activation model involving a thioredoxin-mediated disulfide isomerization at the amino terminus is proposed.NADP-dependent malate dehydrogenase (NADP-MDH) 1 (EC 1.1.1.82) catalyzes the reduction of oxaloacetate into malate in higher plants. In C 4 plants, such as sorghum or maize, it is located in the chloroplasts of mesophyll cells where it participates in the exportation of reducing equivalents needed for the photosynthetic fixation of atmospheric CO 2 into organic molecules in bundle sheath cell chloroplasts (1). Among all the malate dehydrogenases studied so far, the NADP-dependent isoform exhibits a unique property. Whereas MDHs using NAD are permanently active, the NADP-dependent isoform is totally inactive in the dark and activated in the light (2). It is now clearly established that this activation is mediated via the photosynthetic electron transfer and the ferredoxin/thioredoxin system and corresponds to the reduction of disulfides present in the inactive form (3). By thiol derivatization before and after activation and site-directed mutagenesis, the disulfide bridges reduced by thioredoxins have been identified (4, 5). To reach full activity, two disulfide bridges must be reduced: an aminoterminal one (cysteines 24 and 29) and a carboxyl-terminal one (cysteines 365 and 377). When these four cysteines are mutated, the mutant protein is permanently active. The two regulatory disulfide bridges belong to two sequence extensio...

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Section: Table III Kinetic Parameters Of Mutant Nadp-mdhsmentioning

confidence: 85%

An Internal Cysteine Is Involved in the Thioredoxin-dependent Activation of Sorghum Leaf NADP-malate Dehydrogenase

Ruelland

Lemaire-Chamley

Maréchal

et al. 1997

Journal of Biological Chemistry

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“…The standard redox potential of PDI is about k180 mV, much more oxidizing than that of thioredoxin (k260 mV) but less than that of bacterial DsbA (k100 mV) [58][59][60]. The difference in redox potentials is due mainly to the nature of the two intervening residues of the reactive -Cys-Xaa-Xaa-Cys-sequence [61].…”

Section: Scheme 1 Possible Mechanisms Of Disulphide-bond Formation Rmentioning

confidence: 97%

“…The difference in redox potentials is due mainly to the nature of the two intervening residues of the reactive -Cys-Xaa-Xaa-Cys-sequence [61]. PDI is about 50-fold more active than thioredoxin at catalysing the isomerization of disulphide bonds in scrambled RNase [58]. Studies on bacterial thioredoxin in which the thioredoxin-box -Cys-Gly-Pro-Cys-was mutated to -Cys-Gly-His-Cys-resulted in a protein with 10-fold higher oxidizing and disulphide-isomerase activity [8,62] and which had gained the ability to complement S. cere isiae PDI1 null mutants (see below) [38], highlighting the importance of these residues.…”

Section: Scheme 1 Possible Mechanisms Of Disulphide-bond Formation Rmentioning

confidence: 99%

The protein disulphide-isomerase family: unravelling a string of folds

Ferrari

Söling

1999

Biochemical Journal

406

359

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The mammalian protein disulphide-isomerase (PDI) family encompasses several highly divergent proteins that are involved in the processing and maturation of secretory proteins in the endoplasmic reticulum. These proteins are characterized by the presence of one or more domains of roughly 95-110 amino acids related to the cytoplasmic protein thioredoxin. All but the PDI-D subfamily are composed entirely of repeats of such domains, with at least one domain containing and one domain lacking a redox-active -Cys-Xaa-Xaa-Cys-tetrapeptide. In addition to their known roles as redox catalysts and isomerases, the last few years have revealed additional functions of the PDI proteins,

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“…Reduced thioredoxin (Trx) 2 cleaves the disulfide bond, "turns on or off a redox switch," and facilitates protein function or activates an enzyme that results from possible conformational changes (1)(2)(3)(4)(5)(6)(7)(8). These disulfide bonds are generally "intrasubunit or intramolecular disulfide bonds."…”

mentioning

confidence: 99%

Thioredoxin-dependent Enzymatic Activation of Mercaptopyruvate Sulfurtransferase

Nagahara

Yoshii

Abe

et al. 2007

Journal of Biological Chemistry

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Redox-active disulfide bonds have an important role in regulating protein function and enzymatic activity. Reduced thioredoxin (Trx) 2 cleaves the disulfide bond, "turns on or off a redox switch," and facilitates protein function or activates an enzyme that results from possible conformational changes (1-8). These disulfide bonds are generally "intrasubunit or intramolecular disulfide bonds." When these cysteines are adjacent, the redox change most effectively turns the redox switch on and off (9). As an exceptional case, hetero-oligomer ATP synthase is inhibited via the formation of an intersubunit disulfide bond between the bcЈ and ␥ subunits because of a mechanical standstill of the molecular motor (10). This redox switch directly regulates enzymatic activity and can therefore be categorized as a direct and mechanical function. In the cases in which protein function is facilitated via a conformational change caused by cleavage of a disulfide bond, the redox switch is categorized as an indirect function.Rat 3-mercaptopyruvate sulfurtransferase (MST) (EC 2.8

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Comparison of the activities of protein disulphide-isomerase and thioredoxin in catalysing disulphide isomerization in a protein substrate

Cited by 112 publications

References 39 publications

An Internal Cysteine Is Involved in the Thioredoxin-dependent Activation of Sorghum Leaf NADP-malate Dehydrogenase

An Internal Cysteine Is Involved in the Thioredoxin-dependent Activation of Sorghum Leaf NADP-malate Dehydrogenase

The protein disulphide-isomerase family: unravelling a string of folds

Thioredoxin-dependent Enzymatic Activation of Mercaptopyruvate Sulfurtransferase

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