The Reaction of the Soybean Cotyledon Mitochondrial Cyanide-resistant Oxidase with Sulfhydryl Reagents Suggests That α-Keto Acid Activation Involves the Formation of a Thiohemiacetal

Umbach, Ann L.; Siedow, James N.

doi:10.1074/jbc.271.40.25019

Cited by 81 publications

(61 citation statements)

References 45 publications

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“…The more N-terminal cysteine residue highly conserved in higher plants (C 128 of S. guttatum and Arabidopsis thaliana sequences in Fig. 2) could possibly be the site of this redox regulation (Umbach and Siedow 1996). The other cysteine present in all higher plant AOX amino acid sequences (C 203 in Fig.…”

Section: Discussionmentioning

confidence: 99%

Characterization of two genes encoding the mitochondrial alternative oxidase in Chlamydomonas reinhardtii

Dinant¹,

Baurain²,

Coosemans³

et al. 2001

Current Genetics

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Two cDNA clones (AOX1 and AOX2) and the corresponding genes encoding the alternative oxidases (AOXs) from Chlamydomonas reinhardtii were isolated and sequenced. The cDNAs, AOX1 and AOX2, contained open reading frames (ORFs) encoding putative proteins of 360 amino acids and 347 amino acids, respectively. For each of the ORFs, a potential mitochondrial-targeting sequence was found in the 5'-end regions. In comparison to AOX enzymes from plants and fungi, the predicted amino acid sequences of the ORFs showed their highest degree of identity with proteins from Aspergillus niger (38.1% and 37.2%) and Ajellomyces capsulatus (37% and 34.9%). Several residues supposed either to be Fe ligands or to be involved in the ubiquinol-binding site were fully conserved in both C. reinhardtii putative AOX proteins. In contrast, a cysteine residue conserved in the sequences of all higher plants and probably involved in the regulation of the enzyme activity was missing both from the AOX1 and AOX2 amino acid sequences and from protein sequences from various other microorganisms. The transcriptional expression of the AOX1 and AOX2 genes in wild-type cells and in mutant cells deficient in mitochondrial complex III activity was also investigated.

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Section: Discussionmentioning

confidence: 99%

Characterization of two genes encoding the mitochondrial alternative oxidase in Chlamydomonas reinhardtii

Dinant¹,

Baurain²,

Coosemans³

et al. 2001

Current Genetics

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“…A study with soybean suggested that the activation of AOX by pyruvate was due to its interaction with a Cys sulfhydryl to form a thiohemiacetal because activation was mimicked by iodoacetate (Umbach and Siedow, 1996). Since evidence suggests that pyruvate activation takes place from within the mitochondrial matrix (Day et al, 1994;Millar et al, 1996), there are two potential Cys residues for such an interaction (Umbach and Siedow, 1993;Vanlerberghe and McIntosh, 1997).…”

Section: Discussionmentioning

confidence: 99%

“…Significant AOX activity in isolated tobacco mitochondria is dependent upon both reduction of the regulatory disulfide bond and the presence of pyruvate (Vanlerberghe et al, 1995). Studies on soybean AOX suggest that pyruvate action is due to its interaction with a Cys sulfhydryl to form a thiohemiacetal, since the activation is mimicked by iodoacetate (Umbach and Siedow, 1996). It has also been shown that pyruvate acts to increase the V max of AOX without any significant effect on its affinity for ubiquinol (Hoefnagel et al, 1997;Millar et al, 1997).…”

mentioning

confidence: 99%

In Organello and in Vivo Evidence of the Importance of the Regulatory Sulfhydryl/Disulfide System and Pyruvate for Alternative Oxidase Activity in Tobacco

1999

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After isolation of tobacco (Nicotiana tabacum) leaf mitochondria, alternative oxidase (AOX) is predominantly present as the disulfide-linked, less-active "oxidized" form. In an in organello assay, significant AOX activity was dependent upon both the reduction of the regulatory disulfide bond (such as occurs by dithiothreitol) and upon the presence of the activator pyruvate. However, AOX activity in these assays was substantially affected when mitochondria were isolated in the presence of pyruvate. First, pyruvate protects against the oxidation of the regulatory sulfhydryl during isolation, such that subsequent in organello AOX activity is not dependent upon dithiothreitol. Second, pyruvate stabilizes AOX activity, such that mitochondria kept in the presence of pyruvate have higher maximum rates of AOX activity than mitochondria kept for some time in the absence of pyruvate. The ability of pyruvate to protect against AOX oxidation was exploited to assess the in vivo status of the regulatory sulfhydryl/disulfide system. In both tobacco suspension cells and tobacco leaves with high levels of AOX protein, the protein is predominantly present as the "reduced" active form in vivo under a range of respiratory conditions. Experiments also indicate that, while the presence of reduced protein may be a necessary prerequisite for significant AOX activity, it is not sufficient for activity and other factors must also be critical.

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“…It is concluded that the desalted extract contained some pyruvate bound to the enzyme that is released during turnover. Pyruvate may be bound to the enzyme to form a thiohemiacetal, as suggested by Umbach and Siedow (1996). We would like to suggest that pyruvate (or any other effective acid) is a prerequisite for maintaining enzymic activity.…”

Section: Pyruvate Lnvolved In Reaction Mechanismmentioning

confidence: 99%

Substrate Kinetics of the Plant Mitochondrial Alternative Oxidase and the Effects of Pyruvate

et al. 1997

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The kinetics of alternative oxidase (AOX) of Arum ifalicum spadices and soybean (Clycine max L.) cotyledons were studied both with intact mitochondria and with a solubilized, partially purified enzyme. Ubiquinone analogs were screened for their suitability as substrates and ubiquinol-1 was found to be most suitable. The kinetics of ubiquinol-1 oxidation via A O X i n both systems followed Michaelis-Menten kinetics, suggesting that the reaction is limited by a single-step substrate reaction. The kinetics are quite different from those previously described, in which the redox state of ubiquinone-10 was monitored and an increase in substrate was accompanied by a decrease in product. The difference between the systems is discussed. Pyruvate is a potent activator of the enzyme and its presence is essential for maximum activity. The addition of pyruvate to the solubilized enzyme increased the maximum initial velocity from 6.2 f 1.3 to 16.9 & 2.8 pmol O, mg-' protein min-' but had little effect on the Michaelis constant for ubiquinol-1, an analog of ubiquinol, which changed from 11 6 2 73 to 157 f 68 p~.It is concluded that pyruvate (and presumably other keto acids) increases the activity of A O X but does not increase its affinity for its substrate. I n agreement with this is the finding that removal of pyruvate (using lactate dehydrogenase and N A D H ) leads to an 80 to 90% decrease in the reaction rate, suggesting that pyruvate is important in the mechanism of reaction of AOX. The removal of pyruvate from the enzyme required turnover, suggesting that pyruvate is bound to the enzyme and is released during turnover.

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The Reaction of the Soybean Cotyledon Mitochondrial Cyanide-resistant Oxidase with Sulfhydryl Reagents Suggests That α-Keto Acid Activation Involves the Formation of a Thiohemiacetal

Cited by 81 publications

References 45 publications

Characterization of two genes encoding the mitochondrial alternative oxidase in Chlamydomonas reinhardtii

Characterization of two genes encoding the mitochondrial alternative oxidase in Chlamydomonas reinhardtii

In Organello and in Vivo Evidence of the Importance of the Regulatory Sulfhydryl/Disulfide System and Pyruvate for Alternative Oxidase Activity in Tobacco

Substrate Kinetics of the Plant Mitochondrial Alternative Oxidase and the Effects of Pyruvate

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