Role of arginine 177 in the MnII binding site of manganese peroxidase. Studies with R177D, R177E, R177N, and R177Q mutants.
Identifieur interne : 000A69 ( Main/Exploration ); précédent : 000A68; suivant : 000A70Role of arginine 177 in the MnII binding site of manganese peroxidase. Studies with R177D, R177E, R177N, and R177Q mutants.
Auteurs : M D Gelpke [États-Unis] ; H L Youngs ; M H GoldSource :
- European journal of biochemistry [ 0014-2956 ] ; 2000.
Descripteurs français
- KwdFr :
- Arginine (métabolisme), Cinétique (MeSH), Manganèse (métabolisme), Peroxidases (composition chimique), Peroxidases (génétique), Peroxidases (métabolisme), Phanerochaete (enzymologie), Protéines recombinantes (composition chimique), Protéines recombinantes (génétique), Protéines recombinantes (métabolisme), Sites de fixation (MeSH).
- MESH :
- composition chimique : Peroxidases, Protéines recombinantes.
- enzymologie : Phanerochaete.
- génétique : Peroxidases, Protéines recombinantes.
- métabolisme : Arginine, Manganèse, Peroxidases, Protéines recombinantes.
- Cinétique, Sites de fixation.
English descriptors
- KwdEn :
- MESH :
- chemical , chemistry : Peroxidases, Recombinant Proteins.
- chemical , genetics : Peroxidases, Recombinant Proteins.
- chemical , metabolism : Arginine, Manganese, Peroxidases, Recombinant Proteins.
- enzymology : Phanerochaete.
- Binding Sites, Kinetics.
Abstract
Previously, we reported that Arg177 is involved in MnII binding at the MnII binding site of manganese peroxidase isozyme 1 (MnP1) of Phanerochaete chrysosporium by examining two mutants: R177A and R177K. We now report on additional mutants: R177D, R177E, R177N, and R177Q. These new mutant enzymes were produced by homologous expression in P. chrysosporium and were purified to homogeneity. The molecular mass and the UV/visible spectra of the ferric and oxidized intermediates of the mutant enzymes were similar to those of the wild-type enzyme, suggesting proper folding, heme insertion, and preservation of the heme environment. However, steady-state and transient-state kinetic analyses demonstrate significantly altered characteristics of MnII oxidation by these new mutant enzymes. Increased dissociation constants (Kd) and apparent Km values for MnII suggest that these mutations at Arg177 decrease binding of MnII to the enzyme. These lowered binding efficiencies, as observed with the R177A and R177K mutants, suggest that the salt-bridge between Arg177 and the MnII binding ligand Glu35 is disrupted in these new mutants. Decreased kcat values for MnII oxidation, decreased second-order rate constants for compound I reduction (k2app), and decreased first-order rate constants for compound II reduction (k3) indicate that these new mutations also decrease the electron-transfer rate. This decrease in rate constants for compounds I and II reduction was not observed in our previous study on the R177A and R177K mutations. The lower rate constants suggest that, even with high MnII concentrations, the MnII binding geometries may be altered in the MnII binding site of these new mutants. These new results, combined with the results from our previous study, clearly indicate a role for Arg177 in promoting efficient MnII binding and oxidation by MnP.
DOI: 10.1046/j.1432-1327.2000.01798.x
PubMed: 11106414
Affiliations:
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Studies with R177D, R177E, R177N, and R177Q mutants.</title><author><name sortKey="Gelpke, M D" sort="Gelpke, M D" uniqKey="Gelpke M" first="M D" last="Gelpke">M D Gelpke</name><affiliation wicri:level="2"><nlm:affiliation>Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, Beaverton, OR 97006-8921, USA.</nlm:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, Beaverton, OR 97006-8921</wicri:regionArea><placeName><region type="state">Oregon</region></placeName></affiliation></author><author><name sortKey="Youngs, H L" sort="Youngs, H L" uniqKey="Youngs H" first="H L" last="Youngs">H L Youngs</name></author><author><name sortKey="Gold, M H" sort="Gold, M H" uniqKey="Gold M" first="M H" last="Gold">M H Gold</name></author></titleStmt><publicationStmt><idno type="wicri:source">PubMed</idno><date when="2000">2000</date><idno type="RBID">pubmed:11106414</idno><idno type="pmid">11106414</idno><idno type="doi">10.1046/j.1432-1327.2000.01798.x</idno><idno type="wicri:Area/Main/Corpus">000A76</idno><idno type="wicri:explorRef" wicri:stream="Main" wicri:step="Corpus" wicri:corpus="PubMed">000A76</idno><idno type="wicri:Area/Main/Curation">000A76</idno><idno type="wicri:explorRef" wicri:stream="Main" wicri:step="Curation">000A76</idno><idno type="wicri:Area/Main/Exploration">000A76</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en">Role of arginine 177 in the MnII binding site of manganese peroxidase. Studies with R177D, R177E, R177N, and R177Q mutants.</title><author><name sortKey="Gelpke, M D" sort="Gelpke, M D" uniqKey="Gelpke M" first="M D" last="Gelpke">M D Gelpke</name><affiliation wicri:level="2"><nlm:affiliation>Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, Beaverton, OR 97006-8921, USA.</nlm:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, Beaverton, OR 97006-8921</wicri:regionArea><placeName><region type="state">Oregon</region></placeName></affiliation></author><author><name sortKey="Youngs, H L" sort="Youngs, H L" uniqKey="Youngs H" first="H L" last="Youngs">H L Youngs</name></author><author><name sortKey="Gold, M H" sort="Gold, M H" uniqKey="Gold M" first="M H" last="Gold">M H Gold</name></author></analytic><series><title level="j">European journal of biochemistry</title><idno type="ISSN">0014-2956</idno><imprint><date when="2000" type="published">2000</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Arginine (metabolism)</term><term>Binding Sites (MeSH)</term><term>Kinetics (MeSH)</term><term>Manganese (metabolism)</term><term>Peroxidases (chemistry)</term><term>Peroxidases (genetics)</term><term>Peroxidases (metabolism)</term><term>Phanerochaete (enzymology)</term><term>Recombinant Proteins (chemistry)</term><term>Recombinant Proteins (genetics)</term><term>Recombinant Proteins (metabolism)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>Arginine (métabolisme)</term><term>Cinétique (MeSH)</term><term>Manganèse (métabolisme)</term><term>Peroxidases (composition chimique)</term><term>Peroxidases (génétique)</term><term>Peroxidases (métabolisme)</term><term>Phanerochaete (enzymologie)</term><term>Protéines recombinantes (composition chimique)</term><term>Protéines recombinantes (génétique)</term><term>Protéines recombinantes (métabolisme)</term><term>Sites de fixation (MeSH)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>Peroxidases</term><term>Recombinant Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>Peroxidases</term><term>Recombinant Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>Arginine</term><term>Manganese</term><term>Peroxidases</term><term>Recombinant Proteins</term></keywords><keywords scheme="MESH" qualifier="composition chimique" xml:lang="fr"><term>Peroxidases</term><term>Protéines recombinantes</term></keywords><keywords scheme="MESH" qualifier="enzymologie" xml:lang="fr"><term>Phanerochaete</term></keywords><keywords scheme="MESH" qualifier="enzymology" xml:lang="en"><term>Phanerochaete</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Peroxidases</term><term>Protéines recombinantes</term></keywords><keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>Arginine</term><term>Manganèse</term><term>Peroxidases</term><term>Protéines recombinantes</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Binding Sites</term><term>Kinetics</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Cinétique</term><term>Sites de fixation</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Previously, we reported that Arg177 is involved in MnII binding at the MnII binding site of manganese peroxidase isozyme 1 (MnP1) of Phanerochaete chrysosporium by examining two mutants: R177A and R177K. We now report on additional mutants: R177D, R177E, R177N, and R177Q. These new mutant enzymes were produced by homologous expression in P. chrysosporium and were purified to homogeneity. The molecular mass and the UV/visible spectra of the ferric and oxidized intermediates of the mutant enzymes were similar to those of the wild-type enzyme, suggesting proper folding, heme insertion, and preservation of the heme environment. However, steady-state and transient-state kinetic analyses demonstrate significantly altered characteristics of MnII oxidation by these new mutant enzymes. Increased dissociation constants (Kd) and apparent Km values for MnII suggest that these mutations at Arg177 decrease binding of MnII to the enzyme. These lowered binding efficiencies, as observed with the R177A and R177K mutants, suggest that the salt-bridge between Arg177 and the MnII binding ligand Glu35 is disrupted in these new mutants. Decreased kcat values for MnII oxidation, decreased second-order rate constants for compound I reduction (k2app), and decreased first-order rate constants for compound II reduction (k3) indicate that these new mutations also decrease the electron-transfer rate. This decrease in rate constants for compounds I and II reduction was not observed in our previous study on the R177A and R177K mutations. The lower rate constants suggest that, even with high MnII concentrations, the MnII binding geometries may be altered in the MnII binding site of these new mutants. These new results, combined with the results from our previous study, clearly indicate a role for Arg177 in promoting efficient MnII binding and oxidation by MnP.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">11106414</PMID><DateCompleted><Year>2001</Year><Month>01</Month><Day>18</Day></DateCompleted><DateRevised><Year>2019</Year><Month>06</Month><Day>20</Day></DateRevised><Article PubModel="Print"><Journal><ISSN IssnType="Print">0014-2956</ISSN><JournalIssue CitedMedium="Print"><Volume>267</Volume><Issue>24</Issue><PubDate><Year>2000</Year><Month>Dec</Month></PubDate></JournalIssue><Title>European journal of biochemistry</Title><ISOAbbreviation>Eur J Biochem</ISOAbbreviation></Journal><ArticleTitle>Role of arginine 177 in the MnII binding site of manganese peroxidase. Studies with R177D, R177E, R177N, and R177Q mutants.</ArticleTitle><Pagination><MedlinePgn>7038-45</MedlinePgn></Pagination><Abstract><AbstractText>Previously, we reported that Arg177 is involved in MnII binding at the MnII binding site of manganese peroxidase isozyme 1 (MnP1) of Phanerochaete chrysosporium by examining two mutants: R177A and R177K. We now report on additional mutants: R177D, R177E, R177N, and R177Q. These new mutant enzymes were produced by homologous expression in P. chrysosporium and were purified to homogeneity. The molecular mass and the UV/visible spectra of the ferric and oxidized intermediates of the mutant enzymes were similar to those of the wild-type enzyme, suggesting proper folding, heme insertion, and preservation of the heme environment. However, steady-state and transient-state kinetic analyses demonstrate significantly altered characteristics of MnII oxidation by these new mutant enzymes. Increased dissociation constants (Kd) and apparent Km values for MnII suggest that these mutations at Arg177 decrease binding of MnII to the enzyme. These lowered binding efficiencies, as observed with the R177A and R177K mutants, suggest that the salt-bridge between Arg177 and the MnII binding ligand Glu35 is disrupted in these new mutants. Decreased kcat values for MnII oxidation, decreased second-order rate constants for compound I reduction (k2app), and decreased first-order rate constants for compound II reduction (k3) indicate that these new mutations also decrease the electron-transfer rate. This decrease in rate constants for compounds I and II reduction was not observed in our previous study on the R177A and R177K mutations. The lower rate constants suggest that, even with high MnII concentrations, the MnII binding geometries may be altered in the MnII binding site of these new mutants. These new results, combined with the results from our previous study, clearly indicate a role for Arg177 in promoting efficient MnII binding and oxidation by MnP.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Gelpke</LastName><ForeName>M D</ForeName><Initials>MD</Initials><AffiliationInfo><Affiliation>Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, Beaverton, OR 97006-8921, USA.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Youngs</LastName><ForeName>H L</ForeName><Initials>HL</Initials></Author><Author ValidYN="Y"><LastName>Gold</LastName><ForeName>M H</ForeName><Initials>MH</Initials></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType><PublicationType UI="D013486">Research Support, U.S. Gov't, Non-P.H.S.</PublicationType></PublicationTypeList></Article><MedlineJournalInfo><Country>England</Country><MedlineTA>Eur J Biochem</MedlineTA><NlmUniqueID>0107600</NlmUniqueID><ISSNLinking>0014-2956</ISSNLinking></MedlineJournalInfo><ChemicalList><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D011994">Recombinant Proteins</NameOfSubstance></Chemical><Chemical><RegistryNumber>42Z2K6ZL8P</RegistryNumber><NameOfSubstance UI="D008345">Manganese</NameOfSubstance></Chemical><Chemical><RegistryNumber>94ZLA3W45F</RegistryNumber><NameOfSubstance UI="D001120">Arginine</NameOfSubstance></Chemical><Chemical><RegistryNumber>EC 1.11.1.-</RegistryNumber><NameOfSubstance UI="D010544">Peroxidases</NameOfSubstance></Chemical><Chemical><RegistryNumber>EC 1.11.1.13</RegistryNumber><NameOfSubstance UI="C051129">manganese peroxidase</NameOfSubstance></Chemical></ChemicalList><CitationSubset>IM</CitationSubset><MeshHeadingList><MeshHeading><DescriptorName UI="D001120" MajorTopicYN="N">Arginine</DescriptorName><QualifierName UI="Q000378" MajorTopicYN="Y">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D001665" MajorTopicYN="N">Binding Sites</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D007700" MajorTopicYN="N">Kinetics</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D008345" MajorTopicYN="N">Manganese</DescriptorName><QualifierName UI="Q000378" MajorTopicYN="Y">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D010544" MajorTopicYN="N">Peroxidases</DescriptorName><QualifierName UI="Q000737" MajorTopicYN="N">chemistry</QualifierName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName><QualifierName UI="Q000378" MajorTopicYN="Y">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D020075" MajorTopicYN="N">Phanerochaete</DescriptorName><QualifierName UI="Q000201" MajorTopicYN="N">enzymology</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D011994" MajorTopicYN="N">Recombinant Proteins</DescriptorName><QualifierName UI="Q000737" MajorTopicYN="N">chemistry</QualifierName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName><QualifierName UI="Q000378" MajorTopicYN="N">metabolism</QualifierName></MeshHeading></MeshHeadingList></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="pubmed"><Year>2000</Year><Month>12</Month><Day>6</Day><Hour>11</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="medline"><Year>2001</Year><Month>2</Month><Day>28</Day><Hour>10</Hour><Minute>1</Minute></PubMedPubDate><PubMedPubDate PubStatus="entrez"><Year>2000</Year><Month>12</Month><Day>6</Day><Hour>11</Hour><Minute>0</Minute></PubMedPubDate></History><PublicationStatus>ppublish</PublicationStatus><ArticleIdList><ArticleId IdType="pubmed">11106414</ArticleId><ArticleId IdType="pii">ejb1798</ArticleId><ArticleId IdType="doi">10.1046/j.1432-1327.2000.01798.x</ArticleId></ArticleIdList></PubmedData></pubmed><affiliations><list><country><li>États-Unis</li></country><region><li>Oregon</li></region></list><tree><noCountry><name sortKey="Gold, M H" sort="Gold, M H" uniqKey="Gold M" first="M H" last="Gold">M H Gold</name><name sortKey="Youngs, H L" sort="Youngs, H L" uniqKey="Youngs H" first="H L" last="Youngs">H L Youngs</name></noCountry><country name="États-Unis"><region name="Oregon"><name sortKey="Gelpke, M D" sort="Gelpke, M D" uniqKey="Gelpke M" first="M D" last="Gelpke">M D Gelpke</name></region></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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