Steady-state kinetic mechanism of recombinant avocado ACC oxidase: initial velocity and inhibitor studies.
Identifieur interne : 000426 ( Main/Exploration ); précédent : 000425; suivant : 000427Steady-state kinetic mechanism of recombinant avocado ACC oxidase: initial velocity and inhibitor studies.
Auteurs : N M Brunhuber [États-Unis] ; J L Mort ; R E Christoffersen ; N O ReichSource :
- Biochemistry [ 0006-2960 ] ; 2000.
Descripteurs français
- KwdFr :
- Activation enzymatique (génétique), Amino-acid oxidoreductases (antagonistes et inhibiteurs), Amino-acid oxidoreductases (composition chimique), Amino-acid oxidoreductases (génétique), Amino-acid oxidoreductases (isolement et purification), Antienzymes (composition chimique), Cinétique (MeSH), Fixation compétitive (génétique), Lauraceae (enzymologie), Lauraceae (génétique), Protéines recombinantes (antagonistes et inhibiteurs), Protéines recombinantes (biosynthèse), Protéines recombinantes (composition chimique), Protéines recombinantes (isolement et purification), Protéines végétales (antagonistes et inhibiteurs), Protéines végétales (composition chimique), Protéines végétales (isolement et purification), Spécificité du substrat (génétique).
- MESH :
- antagonistes et inhibiteurs : Amino-acid oxidoreductases, Protéines recombinantes, Protéines végétales.
- biosynthèse : Protéines recombinantes.
- composition chimique : Amino-acid oxidoreductases, Antienzymes, Protéines recombinantes, Protéines végétales.
- enzymologie : Lauraceae.
- génétique : Activation enzymatique, Amino-acid oxidoreductases, Fixation compétitive, Lauraceae, Spécificité du substrat.
- isolement et purification : Amino-acid oxidoreductases, Protéines recombinantes, Protéines végétales.
- Cinétique.
English descriptors
- KwdEn :
- Amino Acid Oxidoreductases (antagonists & inhibitors), Amino Acid Oxidoreductases (chemistry), Amino Acid Oxidoreductases (genetics), Amino Acid Oxidoreductases (isolation & purification), Binding, Competitive (genetics), Enzyme Activation (genetics), Enzyme Inhibitors (chemistry), Kinetics (MeSH), Lauraceae (enzymology), Lauraceae (genetics), Plant Proteins (antagonists & inhibitors), Plant Proteins (chemistry), Plant Proteins (isolation & purification), Recombinant Proteins (antagonists & inhibitors), Recombinant Proteins (biosynthesis), Recombinant Proteins (chemistry), Recombinant Proteins (isolation & purification), Substrate Specificity (genetics).
- MESH :
- chemical , antagonists & inhibitors : Amino Acid Oxidoreductases, Plant Proteins, Recombinant Proteins.
- chemical , biosynthesis : Recombinant Proteins.
- chemical , chemistry : Amino Acid Oxidoreductases, Enzyme Inhibitors, Plant Proteins, Recombinant Proteins.
- chemical , genetics : Amino Acid Oxidoreductases.
- chemical , isolation & purification : Amino Acid Oxidoreductases, Plant Proteins, Recombinant Proteins.
- enzymology : Lauraceae.
- genetics : Binding, Competitive, Enzyme Activation, Lauraceae, Substrate Specificity.
- Kinetics.
Abstract
The gaseous plant hormone ethylene modulates a wide range of biological processes, including fruit ripening. It is synthesized by the ascorbate-dependent oxidation of 1-aminocyclopropyl-1-carboxylate (ACC), a reaction catalyzed by ACC oxidase. Recombinant avocado (Persea americana) ACC oxidase was expressed in Escherichia coli and purified in milligram quantities, resulting in high levels of ACC oxidase protein and enzyme activity. An optimized assay for the purified enzyme was developed that takes into account the inherent complexities of the assay system. Fe(II) and ascorbic acid form a binary complex that is not the true substrate for the reaction and enhances the degree of ascorbic acid substrate inhibition. The K(d) value for Fe(II) (40 nM, free species) and the K(m)'s for ascorbic acid (2.1 mM), ACC (62 microM), and O(2) (4 microM) were determined. Fe(II) and ACC exhibit substrate inhibition, and a second metal binding site is suggested. Initial velocity measurements and inhibitor studies were used to resolve the kinetic mechanism through the final substrate binding step. Fe(II) binding is followed by either ascorbate or ACC binding, with ascorbate being preferred. This is followed by the ordered addition of molecular oxygen and the last substrate, leading to the formation of the catalytically competent complex. Both Fe(II) and O(2) are in thermodynamic equilibrium with their enzyme forms. The binding of a second molecule of ascorbic acid or ACC leads to significant substrate inhibition. ACC and ascorbate analogues were used to confirm the kinetic mechanism and to identify important determinants of substrate binding.
DOI: 10.1021/bi0000162
PubMed: 10978157
Affiliations:
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Le document en format XML
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It is synthesized by the ascorbate-dependent oxidation of 1-aminocyclopropyl-1-carboxylate (ACC), a reaction catalyzed by ACC oxidase. Recombinant avocado (Persea americana) ACC oxidase was expressed in Escherichia coli and purified in milligram quantities, resulting in high levels of ACC oxidase protein and enzyme activity. An optimized assay for the purified enzyme was developed that takes into account the inherent complexities of the assay system. Fe(II) and ascorbic acid form a binary complex that is not the true substrate for the reaction and enhances the degree of ascorbic acid substrate inhibition. The K(d) value for Fe(II) (40 nM, free species) and the K(m)'s for ascorbic acid (2.1 mM), ACC (62 microM), and O(2) (4 microM) were determined. Fe(II) and ACC exhibit substrate inhibition, and a second metal binding site is suggested. Initial velocity measurements and inhibitor studies were used to resolve the kinetic mechanism through the final substrate binding step. Fe(II) binding is followed by either ascorbate or ACC binding, with ascorbate being preferred. This is followed by the ordered addition of molecular oxygen and the last substrate, leading to the formation of the catalytically competent complex. Both Fe(II) and O(2) are in thermodynamic equilibrium with their enzyme forms. The binding of a second molecule of ascorbic acid or ACC leads to significant substrate inhibition. ACC and ascorbate analogues were used to confirm the kinetic mechanism and to identify important determinants of substrate binding.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">10978157</PMID><DateCompleted><Year>2000</Year><Month>09</Month><Day>28</Day></DateCompleted><DateRevised><Year>2019</Year><Month>06</Month><Day>13</Day></DateRevised><Article PubModel="Print"><Journal><ISSN IssnType="Print">0006-2960</ISSN><JournalIssue CitedMedium="Print"><Volume>39</Volume><Issue>35</Issue><PubDate><Year>2000</Year><Month>Sep</Month><Day>05</Day></PubDate></JournalIssue><Title>Biochemistry</Title><ISOAbbreviation>Biochemistry</ISOAbbreviation></Journal><ArticleTitle>Steady-state kinetic mechanism of recombinant avocado ACC oxidase: initial velocity and inhibitor studies.</ArticleTitle><Pagination><MedlinePgn>10730-8</MedlinePgn></Pagination><Abstract><AbstractText>The gaseous plant hormone ethylene modulates a wide range of biological processes, including fruit ripening. It is synthesized by the ascorbate-dependent oxidation of 1-aminocyclopropyl-1-carboxylate (ACC), a reaction catalyzed by ACC oxidase. Recombinant avocado (Persea americana) ACC oxidase was expressed in Escherichia coli and purified in milligram quantities, resulting in high levels of ACC oxidase protein and enzyme activity. An optimized assay for the purified enzyme was developed that takes into account the inherent complexities of the assay system. Fe(II) and ascorbic acid form a binary complex that is not the true substrate for the reaction and enhances the degree of ascorbic acid substrate inhibition. The K(d) value for Fe(II) (40 nM, free species) and the K(m)'s for ascorbic acid (2.1 mM), ACC (62 microM), and O(2) (4 microM) were determined. Fe(II) and ACC exhibit substrate inhibition, and a second metal binding site is suggested. Initial velocity measurements and inhibitor studies were used to resolve the kinetic mechanism through the final substrate binding step. Fe(II) binding is followed by either ascorbate or ACC binding, with ascorbate being preferred. This is followed by the ordered addition of molecular oxygen and the last substrate, leading to the formation of the catalytically competent complex. Both Fe(II) and O(2) are in thermodynamic equilibrium with their enzyme forms. The binding of a second molecule of ascorbic acid or ACC leads to significant substrate inhibition. ACC and ascorbate analogues were used to confirm the kinetic mechanism and to identify important determinants of substrate binding.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Brunhuber</LastName><ForeName>N M</ForeName><Initials>NM</Initials><AffiliationInfo><Affiliation>Department of Chemistry and Biochemistry and Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara, Santa Barbara, California 93106, USA.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Mort</LastName><ForeName>J L</ForeName><Initials>JL</Initials></Author><Author ValidYN="Y"><LastName>Christoffersen</LastName><ForeName>R E</ForeName><Initials>RE</Initials></Author><Author ValidYN="Y"><LastName>Reich</LastName><ForeName>N O</ForeName><Initials>NO</Initials></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D003160">Comparative Study</PublicationType><PublicationType UI="D016428">Journal Article</PublicationType><PublicationType UI="D013486">Research Support, U.S. Gov't, Non-P.H.S.</PublicationType></PublicationTypeList></Article><MedlineJournalInfo><Country>United States</Country><MedlineTA>Biochemistry</MedlineTA><NlmUniqueID>0370623</NlmUniqueID><ISSNLinking>0006-2960</ISSNLinking></MedlineJournalInfo><ChemicalList><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D004791">Enzyme Inhibitors</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D010940">Plant Proteins</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D011994">Recombinant Proteins</NameOfSubstance></Chemical><Chemical><RegistryNumber>EC 1.4.-</RegistryNumber><NameOfSubstance UI="D000594">Amino Acid Oxidoreductases</NameOfSubstance></Chemical><Chemical><RegistryNumber>EC 1.4.3.-</RegistryNumber><NameOfSubstance UI="C059460">1-aminocyclopropane-1-carboxylic acid oxidase</NameOfSubstance></Chemical></ChemicalList><CitationSubset>IM</CitationSubset><MeshHeadingList><MeshHeading><DescriptorName UI="D000594" MajorTopicYN="N">Amino Acid Oxidoreductases</DescriptorName><QualifierName UI="Q000037" MajorTopicYN="Y">antagonists & inhibitors</QualifierName><QualifierName UI="Q000737" MajorTopicYN="Y">chemistry</QualifierName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName><QualifierName UI="Q000302" MajorTopicYN="N">isolation & purification</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D001667" MajorTopicYN="N">Binding, Competitive</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D004789" MajorTopicYN="N">Enzyme Activation</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D004791" MajorTopicYN="N">Enzyme Inhibitors</DescriptorName><QualifierName UI="Q000737" MajorTopicYN="Y">chemistry</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D007700" MajorTopicYN="N">Kinetics</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D019770" MajorTopicYN="N">Lauraceae</DescriptorName><QualifierName UI="Q000201" MajorTopicYN="Y">enzymology</QualifierName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D010940" MajorTopicYN="N">Plant Proteins</DescriptorName><QualifierName UI="Q000037" MajorTopicYN="N">antagonists & inhibitors</QualifierName><QualifierName UI="Q000737" MajorTopicYN="N">chemistry</QualifierName><QualifierName UI="Q000302" MajorTopicYN="N">isolation & purification</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D011994" MajorTopicYN="N">Recombinant Proteins</DescriptorName><QualifierName UI="Q000037" MajorTopicYN="Y">antagonists & inhibitors</QualifierName><QualifierName UI="Q000096" MajorTopicYN="N">biosynthesis</QualifierName><QualifierName UI="Q000737" MajorTopicYN="Y">chemistry</QualifierName><QualifierName UI="Q000302" MajorTopicYN="N">isolation & purification</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D013379" MajorTopicYN="N">Substrate Specificity</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName></MeshHeading></MeshHeadingList></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="pubmed"><Year>2000</Year><Month>9</Month><Day>9</Day><Hour>11</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="medline"><Year>2000</Year><Month>9</Month><Day>30</Day><Hour>11</Hour><Minute>1</Minute></PubMedPubDate><PubMedPubDate PubStatus="entrez"><Year>2000</Year><Month>9</Month><Day>9</Day><Hour>11</Hour><Minute>0</Minute></PubMedPubDate></History><PublicationStatus>ppublish</PublicationStatus><ArticleIdList><ArticleId IdType="pubmed">10978157</ArticleId><ArticleId IdType="pii">bi0000162</ArticleId><ArticleId IdType="doi">10.1021/bi0000162</ArticleId></ArticleIdList></PubmedData></pubmed><affiliations><list><country><li>États-Unis</li></country></list><tree><noCountry><name sortKey="Christoffersen, R E" sort="Christoffersen, R E" uniqKey="Christoffersen R" first="R E" last="Christoffersen">R E Christoffersen</name><name sortKey="Mort, J L" sort="Mort, J L" uniqKey="Mort J" first="J L" last="Mort">J L Mort</name><name sortKey="Reich, N O" sort="Reich, N O" uniqKey="Reich N" first="N O" last="Reich">N O Reich</name></noCountry><country name="États-Unis"><noRegion><name sortKey="Brunhuber, N M" sort="Brunhuber, N M" uniqKey="Brunhuber N" first="N M" last="Brunhuber">N M Brunhuber</name></noRegion></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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