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<document id="523FEC7927ED46D0CF339B8E5B898BC6" ID-DOI="10.1016/j.phytochem.2021.112881" ID-GBIF-Dataset="2ea59e53-b059-41a5-b5b5-30cb32d3aaf0" ID-ISSN="1873-3700" ID-Zenodo-Dep="8270251" IM.bibliography_approvedBy="juliana" IM.illustrations_approvedBy="juliana" IM.materialsCitations_approvedBy="felipe" IM.metadata_approvedBy="juliana" IM.tables_approvedBy="juliana" IM.taxonomicNames_approvedBy="juliana" IM.treatments_approvedBy="juliana" checkinTime="1692304096437" checkinUser="felipe" docAuthor="Hýskova, Veronika, Belonozníkov, Katerina, Smeringaiova, Ingrida, Kavan, Daniel, Ingr, Marek &amp; Ryslava, Helena" docDate="2021" docId="0139879D1C4EFFC1FFC9FB0D70D594A4" docLanguage="en" docName="Phytochemistry.190.112881.pdf" docOrigin="Phytochemistry (112881) 190" docSource="http://dx.doi.org/10.1016/j.phytochem.2021.112881" docStyle="DocumentStyle:F36D69FC8B198FBE91029DF9C24697D3.5:Phytochemistry.2020-.journal_article" docStyleId="F36D69FC8B198FBE91029DF9C24697D3" docStyleName="Phytochemistry.2020-.journal_article" docStyleVersion="5" docTitle="Petroselinum crispum subsp. root SDH" docType="treatment" docVersion="6" lastPageNumber="3" masterDocId="FD00FFE51C4FFFC3FFADFFE27321920C" masterDocTitle="How is the activity of shikimate dehydrogenase from the root of Petroselinum crispum (parsley) regulated and which side reactions are catalyzed?" masterLastPageNumber="12" masterPageNumber="1" pageNumber="2" updateTime="1692634466674" updateUser="ExternalLinkService">
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<mods:title id="2C739C15B12691CCED5D061560BB2DF1">How is the activity of shikimate dehydrogenase from the root of Petroselinum crispum (parsley) regulated and which side reactions are catalyzed?</mods:title>
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<mods:namePart id="FCD5AE6AC3390F0475D1F1AA18FD0160">Hýskova, Veronika</mods:namePart>
<mods:affiliation id="CD0B4DB81015D69BF5DD039358A93E2F">Department of Biochemistry, Faculty of Science, Charles University, Hlavova 2030, Prague 2, 128 40, Czech Republic</mods:affiliation>
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<mods:namePart id="F8D07342BDB280851EDE110164B04B1E">Belonozníkov, Katerina</mods:namePart>
<mods:affiliation id="25F62D2AB8C50D445DABA22EF0F5268F">Department of Biochemistry, Faculty of Science, Charles University, Hlavova 2030, Prague 2, 128 40, Czech Republic</mods:affiliation>
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<mods:namePart id="7FAF0A0F9BF7AFCD9F9F78B64C5E016D">Smeringaiova, Ingrida</mods:namePart>
<mods:affiliation id="5C7807FF27EE710E604317C681D0F69C">Department of Biochemistry, Faculty of Science, Charles University, Hlavova 2030, Prague 2, 128 40, Czech Republic</mods:affiliation>
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<mods:namePart id="4CF6AE5B1D70E4972C883FC9D7167916">Kavan, Daniel</mods:namePart>
<mods:affiliation id="39CBFD37AB2E3C844775B5511A5DAFF7">Department of Biochemistry, Faculty of Science, Charles University, Hlavova 2030, Prague 2, 128 40, Czech Republic</mods:affiliation>
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<mods:namePart id="04C61A39F395CA7CE62CA1D33C4ADAB9">Ingr, Marek</mods:namePart>
<mods:affiliation id="9E7A7C9B52E6C2DE4B34A2C76FEE78F4">Department of Biochemistry, Faculty of Science, Charles University, Hlavova 2030, Prague 2, 128 40, Czech Republic &amp; Tomas Bata University in Zlín, Faculty of Technology, Department of Physics and Materials Engineering, N´am. T. G. Masaryka 5555, 760 01, Zlín, Czech Republic &amp; Tomas Bata University in Zlín, Faculty of Technology, Department of Physics and Materials Engineering, N´am. T. G. Masaryka 5555, 760 01, Zlín, Czech Republic</mods:affiliation>
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<mods:namePart id="F71F4EDBEA3D44EC8EC9F7753578FEE3">Ryslava, Helena</mods:namePart>
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<mods:title id="5637632325D3886103C1172108BADCCC">Phytochemistry</mods:title>
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<treatment id="0139879D1C4EFFC1FFC9FB0D70D594A4" ID-DOI="http://doi.org/10.5281/zenodo.8264312" ID-GBIF-Taxon="212439141" ID-Zenodo-Dep="8264312" LSID="urn:lsid:plazi:treatment:0139879D1C4EFFC1FFC9FB0D70D594A4" httpUri="http://treatment.plazi.org/id/0139879D1C4EFFC1FFC9FB0D70D594A4" lastPageId="2" lastPageNumber="3" pageId="1" pageNumber="2">
<subSubSection id="C18A65001C4EFFC2FFC9FB0D72A19712" pageId="1" pageNumber="2" type="nomenclature">
<paragraph id="892F368B1C4EFFC2FFC9FB0D72A19712" blockId="1.[100,747,1263,1310]" pageId="1" pageNumber="2">
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<heading id="D26781E71C4EFFC2FFC9FB0D71CA970E" bold="true" box="[100,747,1263,1283]" fontSize="36" level="1" pageId="1" pageNumber="2" reason="1">
2.1. Characterization of
<taxonomicName id="4E904D081C4EFFC2FEE4FB127286970E" ID-CoL="6V8D2" ID-ENA="4043" authority="SDH" authorityName="SDH" baseAuthorityName="Mill." box="[329,423,1263,1283]" class="Magnoliopsida" family="Apiaceae" genus="Petroselinum" kingdom="Plantae" order="Apiales" pageId="1" pageNumber="2" phylum="Tracheophyta" rank="species" species="crispum">P. crispum</taxonomicName>
root SDH: typical high pH optimum
</heading>
<heading id="D26781E71C4EFFC2FFC9FAE972A19712" box="[100,384,1291,1310]" fontSize="8" level="3" pageId="1" pageNumber="2" reason="8">and 61.5 kDa molecular weight</heading>
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SDH activity was screened in crude extracts from 8 different vegetables. Onion (
<taxonomicName id="4E904D081C4EFFC2FF5CFABD727B977E" box="[241,346,1375,1394]" class="Liliopsida" family="Amaryllidaceae" genus="Allium" kingdom="Plantae" order="Asparagales" pageId="1" pageNumber="2" phylum="Tracheophyta" rank="species" species="cepa">
<emphasis id="BBE4EA991C4EFFC2FF5CFABD727B977E" bold="true" box="[241,346,1375,1394]" italics="true" pageId="1" pageNumber="2">Allium cepa</emphasis>
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L., bulb) and broccoli (
<taxonomicName id="4E904D081C4EFFC2FD91FABD71F7977E" box="[572,726,1375,1394]" class="Magnoliopsida" family="Brassicaceae" genus="Brassica" kingdom="Plantae" order="Brassicales" pageId="1" pageNumber="2" phylum="Tracheophyta" rank="species" species="oleracea">
<emphasis id="BBE4EA991C4EFFC2FD91FABD71F7977E" bold="true" box="[572,726,1375,1394]" italics="true" pageId="1" pageNumber="2">Brassica oleracea</emphasis>
</taxonomicName>
<taxonomicName id="4E904D081C4EFFC2FD73FA8273819782" pageId="1" pageNumber="2" rank="variety" variety="italica">var. italica</taxonomicName>
, florets) crude extracts had low SDH activity, whereas crude extracts from
<taxonomicName id="4E904D081C4EFFC2FF7BFA75724597A6" box="[214,356,1431,1450]" class="Magnoliopsida" family="Apiaceae" genus="Petroselinum" kingdom="Plantae" order="Apiales" pageId="1" pageNumber="8" phylum="Tracheophyta" rank="subSpecies" species="crispum" subSpecies="root">
<emphasis id="BBE4EA991C4EFFC2FF7BFA75721497A6" bold="true" box="[214,309,1431,1450]" italics="true" pageId="1" pageNumber="2">P. crispum</emphasis>
root
</taxonomicName>
and zucchini (
<taxonomicName id="4E904D081C4EFFC2FE5AFA75715F97A6" box="[503,638,1431,1450]" class="Magnoliopsida" family="Cucurbitaceae" genus="Cucurbita" kingdom="Plantae" order="Cucurbitales" pageId="1" pageNumber="2" phylum="Tracheophyta" rank="species" species="pepo">
<emphasis id="BBE4EA991C4EFFC2FE5AFA75715F97A6" bold="true" box="[503,638,1431,1450]" italics="true" pageId="1" pageNumber="2">Cucurbita pepo</emphasis>
</taxonomicName>
L.
<taxonomicName id="4E904D081C4EFFC2FD0DFA7573BD97CA" pageId="1" pageNumber="2" rank="variety" variety="cylindrical">var. cylindrical</taxonomicName>
, pulp) were identified as the richest sources of SDH among all plants tested. A high SDH activity per gram of fresh weight correlates with a low total phenolics content and
<emphasis id="BBE4EA991C4EFFC2FE4EFA08711A97F2" bold="true" box="[483,571,1514,1534]" italics="true" pageId="1" pageNumber="2">vice versa</emphasis>
(
<figureCitation id="11AB2A0E1C4EFFC2FDE6FA0871B797F2" box="[587,662,1514,1534]" captionStart="Fig" captionStartId="2.[100,130,1042,1059]" captionTargetBox="[264,1323,149,1014]" captionTargetId="figure-308@2.[263,1324,148,1015]" captionTargetPageId="2" captionText="Fig. 1. Determination of the type of SDH reaction mechanism based on Lineweaver-Burk diagnostical plots for the direction from SA to DHS (A,B) and from DHS to SA (C,D) Double reciprocal plots are fitted to an equation corresponding to a sequential mechanism." figureDoi="http://doi.org/10.5281/zenodo.8270255" httpUri="https://zenodo.org/record/8270255/files/figure.png" pageId="1" pageNumber="2">Fig. A.1</figureCitation>
). SDH was purified by ion exchange and gel chromatography from
<taxonomicName id="4E904D081C4EFFC2FD32F9E570239415" box="[671,770,1542,1562]" class="Magnoliopsida" family="Apiaceae" genus="Petroselinum" kingdom="Plantae" order="Apiales" pageId="1" pageNumber="2" phylum="Tracheophyta" rank="species" species="crispum">
<emphasis id="BBE4EA991C4EFFC2FD32F9E570239415" bold="true" box="[671,770,1542,1562]" italics="true" pageId="1" pageNumber="2">P. crispum</emphasis>
</taxonomicName>
(
<taxonomicName id="4E904D081C4EFFC2FFC1F9C072089439" box="[108,297,1570,1589]" class="Magnoliopsida" family="Apiaceae" genus="Petroselinum" kingdom="Plantae" order="Apiales" pageId="1" pageNumber="2" phylum="Tracheophyta" rank="species" species="crispum">
<emphasis id="BBE4EA991C4EFFC2FFC1F9C072089439" bold="true" box="[108,297,1570,1589]" italics="true" pageId="1" pageNumber="2">Petroselinum crispum</emphasis>
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) root to a final specific activity of 470 ± 18 nmol. min
<superScript id="7EE59BC31C4EFFC2FF24F9DA73B6944A" attach="left" box="[137,151,1592,1606]" fontSize="6" pageId="1" pageNumber="2">-1</superScript>
.mg
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.
</paragraph>
<paragraph id="892F368B1C4EFFC2FF29F9B872CA9541" blockId="1.[100,771,1347,1869]" pageId="1" pageNumber="2">
SDH activity was pH-dependent, with a pH optimum between 7-8.5 and
<quantity id="4E689B6E1C4EFFC2FF3EF99473CF9485" box="[147,238,1654,1673]" metricMagnitude="-1" metricUnit="m" metricValue="2.4765" metricValueMax="2.54" metricValueMin="2.413" pageId="1" pageNumber="2" unit="in" value="9.75" valueMax="10.0" valueMin="9.5">9.5-10 in</quantity>
the physiological and reverse reactions, respectively (
<tableCitation id="C41203301C4EFFC2FFC1F970739594A9" box="[108,180,1682,1701]" captionStart="Table 1" captionStartId="1.[818,868,150,166]" captionTargetPageId="1" captionText="Table 1 Kinetic parameters of P. crispum SDH. Vmax - maximal reaction rate of enzyme reaction, K - Michaelis constant of the specific substrate, K -apparent m m Michaelis constant at a given concentration of the second substrate), KSS - inhibition constant at an excess substrate concentration, KA - dissociation constant of the enzyme substrate complex, ♣ for technical reasons (absorbance above 3 or excess substrate inhibition), this parameter could not be determined. Kinetic constants were calculated as an average of values from at least 3 experiments performed in doublets, S. D. are shown." httpUri="http://table.plazi.org/id/DDEF66031C4EFFC2FC9FFF7477A5937F" pageId="1" pageNumber="2" tableUuid="DDEF66031C4EFFC2FC9FFF7477A5937F">Table 1</tableCitation>
). The molecular weight was 63 kDa when determined by red native electrophoresis (
<figureCitation id="11AB2A0E1C4EFFC2FEE5F94C728294CD" box="[328,419,1710,1729]" captionStart="Fig" captionStartId="4.[100,130,148,165]" captionTargetId="figure-7@3.[259,1329,148,1837]" captionTargetPageId="3" captionText="Fig. 2. Product inhibition analysis for the determination of the mechanism of bisubstrate reaction in the direction from SA to DHS (A-F) and in the direction from DHS to SA (G-J). Experimental data are fitted with calculated values determined by non-linear regression using equations charactering competitive and non-competitive inhibition, respectively. SDH products DHS (A,B,C) and NADPH (D,E,F) served as competitive (B,C,D,F) and non-competitive (A,E) inhibitors. The saturating (C,F) and subsaturating (A,B,D,E) concentrations of SA and NADP were 20 mM and 2 mM, and 0.3 mM and 0.5 mM, respectively. Saturating concentrations of NADP and variable concentrations of SA caused no inhibition (N.I.) of NADPH and saturating concentrations of SA and variable concentrations of NADP caused N.I. of DHS (data not shown). SDH products SA (G,H) and NADP (I,J) served as competitive (G,I) and non-competitive (H,J) inhibitors at 0.75 mM (subsaturating concentration) DHS (G,I) and 0.2 mM (subsaturating concentration) NADPH (H,J) as a second substrate. Ki indicates inhibition constants in mM. NC - noncompetitive and C - competitive inhibition. Measurements were performed in doublets (S.D. are shown) and at least 2-4 times (enzyme preparations from different isolations)." figureDoi="http://doi.org/10.5281/zenodo.8270257" httpUri="https://zenodo.org/record/8270257/files/figure.png" pageId="1" pageNumber="2">Fig. A.2A</figureCitation>
) and 60 kDa when assessed by gel chromatography (
<figureCitation id="11AB2A0E1C4EFFC2FEA1F928724094D1" box="[268,353,1738,1757]" captionStart="Fig" captionStartId="4.[100,130,148,165]" captionTargetId="figure-7@3.[259,1329,148,1837]" captionTargetPageId="3" captionText="Fig. 2. Product inhibition analysis for the determination of the mechanism of bisubstrate reaction in the direction from SA to DHS (A-F) and in the direction from DHS to SA (G-J). Experimental data are fitted with calculated values determined by non-linear regression using equations charactering competitive and non-competitive inhibition, respectively. SDH products DHS (A,B,C) and NADPH (D,E,F) served as competitive (B,C,D,F) and non-competitive (A,E) inhibitors. The saturating (C,F) and subsaturating (A,B,D,E) concentrations of SA and NADP were 20 mM and 2 mM, and 0.3 mM and 0.5 mM, respectively. Saturating concentrations of NADP and variable concentrations of SA caused no inhibition (N.I.) of NADPH and saturating concentrations of SA and variable concentrations of NADP caused N.I. of DHS (data not shown). SDH products SA (G,H) and NADP (I,J) served as competitive (G,I) and non-competitive (H,J) inhibitors at 0.75 mM (subsaturating concentration) DHS (G,I) and 0.2 mM (subsaturating concentration) NADPH (H,J) as a second substrate. Ki indicates inhibition constants in mM. NC - noncompetitive and C - competitive inhibition. Measurements were performed in doublets (S.D. are shown) and at least 2-4 times (enzyme preparations from different isolations)." figureDoi="http://doi.org/10.5281/zenodo.8270257" httpUri="https://zenodo.org/record/8270257/files/figure.png" pageId="1" pageNumber="2">Fig. A.2B</figureCitation>
). The isoelectric point of
<taxonomicName id="4E904D081C4EFFC2FDE2F92871F894D1" authority="SDH" authorityName="SDH" box="[591,729,1738,1757]" class="Magnoliopsida" family="Apiaceae" genus="Petroselinum" kingdom="Plantae" order="Apiales" pageId="1" pageNumber="2" phylum="Tracheophyta" rank="species" species="crispum">
<emphasis id="BBE4EA991C4EFFC2FDE2F928718B94D1" bold="true" box="[591,682,1738,1757]" italics="true" pageId="1" pageNumber="2">P. crispum</emphasis>
SDH
</taxonomicName>
was 4.5 (
<figureCitation id="11AB2A0E1C4EFFC2FF3CF90473C994F5" box="[145,232,1766,1785]" captionStart="Fig" captionStartId="4.[100,130,148,165]" captionTargetId="figure-7@3.[259,1329,148,1837]" captionTargetPageId="3" captionText="Fig. 2. Product inhibition analysis for the determination of the mechanism of bisubstrate reaction in the direction from SA to DHS (A-F) and in the direction from DHS to SA (G-J). Experimental data are fitted with calculated values determined by non-linear regression using equations charactering competitive and non-competitive inhibition, respectively. SDH products DHS (A,B,C) and NADPH (D,E,F) served as competitive (B,C,D,F) and non-competitive (A,E) inhibitors. The saturating (C,F) and subsaturating (A,B,D,E) concentrations of SA and NADP were 20 mM and 2 mM, and 0.3 mM and 0.5 mM, respectively. Saturating concentrations of NADP and variable concentrations of SA caused no inhibition (N.I.) of NADPH and saturating concentrations of SA and variable concentrations of NADP caused N.I. of DHS (data not shown). SDH products SA (G,H) and NADP (I,J) served as competitive (G,I) and non-competitive (H,J) inhibitors at 0.75 mM (subsaturating concentration) DHS (G,I) and 0.2 mM (subsaturating concentration) NADPH (H,J) as a second substrate. Ki indicates inhibition constants in mM. NC - noncompetitive and C - competitive inhibition. Measurements were performed in doublets (S.D. are shown) and at least 2-4 times (enzyme preparations from different isolations)." figureDoi="http://doi.org/10.5281/zenodo.8270257" httpUri="https://zenodo.org/record/8270257/files/figure.png" pageId="1" pageNumber="2">Fig. A.2C</figureCitation>
). Only one protein band with SDH activity was detected after native red electrophoresis and isoelectric focusing. During gel chromatography, SDH was eluted as a single peak. Thus, only one isoform of SDH is present in
<taxonomicName id="4E904D081C4EFFC2FEF7F8D872C69541" box="[346,487,1849,1869]" class="Magnoliopsida" family="Apiaceae" genus="Petroselinum" kingdom="Plantae" order="Apiales" pageId="1" pageNumber="8" phylum="Tracheophyta" rank="subSpecies" species="crispum" subSpecies="root">
<emphasis id="BBE4EA991C4EFFC2FEF7F8D872969540" bold="true" box="[346,439,1849,1869]" italics="true" pageId="1" pageNumber="2">P. crispum</emphasis>
root
</taxonomicName>
.
</paragraph>
<paragraph id="892F368B1C4EFFC2FC9FFCEF709D9130" blockId="1.[818,1464,781,828]" pageId="1" pageNumber="2">
<emphasis id="BBE4EA991C4EFFC2FC9FFCEF709D9130" bold="true" italics="true" pageId="1" pageNumber="2">
2.2. The kinetic properties of
<taxonomicName id="4E904D081C4EFFC2FBECFCEF77EE912C" authority="SDH" authorityName="SDH" box="[1089,1231,781,800]" class="Magnoliopsida" family="Apiaceae" genus="Petroselinum" kingdom="Plantae" order="Apiales" pageId="1" pageNumber="2" phylum="Tracheophyta" rank="species" species="crispum">P. crispum SDH</taxonomicName>
differ from those of other dehydrogenases
</emphasis>
</paragraph>
<paragraph id="892F368B1C4EFFC2FCFCFC8370AF96AB" blockId="1.[818,1488,865,1191]" pageId="1" pageNumber="2">
The kinetic parameters of the reaction catalyzed by
<taxonomicName id="4E904D081C4EFFC2FAEEFC83707D919C" authority="SDH" authorityName="SDH" class="Magnoliopsida" family="Apiaceae" genus="Petroselinum" kingdom="Plantae" order="Apiales" pageId="1" pageNumber="8" phylum="Tracheophyta" rank="subSpecies" species="crispum" subSpecies="root">
<emphasis id="BBE4EA991C4EFFC2FAEEFC8376809178" bold="true" box="[1347,1441,865,884]" italics="true" pageId="1" pageNumber="2">P. crispum</emphasis>
root SDH
</taxonomicName>
were studied in both directions, i.e., in the physiological (shikimate pathway) direction: NADPH + DHS → SA + NADP, and in the reverse direction: SA + NADP → NADPH + DHS (
<figureCitation id="11AB2A0E1C4EFFC2FB60FC56762591CB" box="[1229,1284,948,967]" captionStart="Fig" captionStartId="2.[100,130,1042,1059]" captionTargetBox="[264,1323,149,1014]" captionTargetId="figure-308@2.[263,1324,148,1015]" captionTargetPageId="2" captionText="Fig. 1. Determination of the type of SDH reaction mechanism based on Lineweaver-Burk diagnostical plots for the direction from SA to DHS (A,B) and from DHS to SA (C,D) Double reciprocal plots are fitted to an equation corresponding to a sequential mechanism." figureDoi="http://doi.org/10.5281/zenodo.8270255" httpUri="https://zenodo.org/record/8270255/files/figure.png" pageId="1" pageNumber="2">Fig. 1</figureCitation>
). The Michaelis constants and maximal velocities are summarized in
<tableCitation id="C41203301C4EFFC2FAAFFC32766991EF" box="[1282,1352,976,995]" captionStart="Table 1" captionStartId="1.[818,868,150,166]" captionTargetPageId="1" captionText="Table 1 Kinetic parameters of P. crispum SDH. Vmax - maximal reaction rate of enzyme reaction, K - Michaelis constant of the specific substrate, K -apparent m m Michaelis constant at a given concentration of the second substrate), KSS - inhibition constant at an excess substrate concentration, KA - dissociation constant of the enzyme substrate complex, ♣ for technical reasons (absorbance above 3 or excess substrate inhibition), this parameter could not be determined. Kinetic constants were calculated as an average of values from at least 3 experiments performed in doublets, S. D. are shown." httpUri="http://table.plazi.org/id/DDEF66031C4EFFC2FC9FFF7477A5937F" pageId="1" pageNumber="2" tableUuid="DDEF66031C4EFFC2FC9FFF7477A5937F">Table 1</tableCitation>
. The maximal reaction rate for the reverse reaction from SA to DHS was 4.6-fold higher than in the shikimate pathway direction (DHS reduction). Because the saturating concentrations of NADP and SA were high, we also determined the apparent Michaelis constant at approximate conditions (
<tableCitation id="C41203301C4EFFC2FC97FBBE70A59663" box="[826,900,1116,1135]" captionStart="Table 1" captionStartId="1.[818,868,150,166]" captionTargetPageId="1" captionText="Table 1 Kinetic parameters of P. crispum SDH. Vmax - maximal reaction rate of enzyme reaction, K - Michaelis constant of the specific substrate, K -apparent m m Michaelis constant at a given concentration of the second substrate), KSS - inhibition constant at an excess substrate concentration, KA - dissociation constant of the enzyme substrate complex, ♣ for technical reasons (absorbance above 3 or excess substrate inhibition), this parameter could not be determined. Kinetic constants were calculated as an average of values from at least 3 experiments performed in doublets, S. D. are shown." httpUri="http://table.plazi.org/id/DDEF66031C4EFFC2FC9FFF7477A5937F" pageId="1" pageNumber="2" tableUuid="DDEF66031C4EFFC2FC9FFF7477A5937F">Table 1</tableCitation>
). Furthermore, excess substrate DHS inhibited the reaction (
<figureCitation id="11AB2A0E1C4EFFC2FC97FB9A70A29687" box="[826,899,1144,1163]" captionStart="Fig" captionStartId="4.[100,130,1529,1546]" captionTargetBox="[345,1246,439,1500]" captionTargetId="figure-625@4.[339,1249,437,1501]" captionTargetPageId="4" captionText="Fig. 3. Proposed kinetic mechanism of the bisubstrate reaction catalyzed by P. crispum SDH from non-photosynthetic tissue. SDH reaction scheme (A), graphical representation of the product inhibition study in DHS reduction direction (B) and in SA oxidation direction (C) with a formation of dead-end complexes. Non-Competitive (NC) and competitive (C) product inhibitors are indicated." figureDoi="http://doi.org/10.5281/zenodo.8270259" httpUri="https://zenodo.org/record/8270259/files/figure.png" pageId="1" pageNumber="2">Fig. A.3</figureCitation>
) with a substrate inhibition constant of KSS = 0.12 ± 0.07 mM (
<tableCitation id="C41203301C4EFFC2FC97FB7170A196AB" box="[826,896,1171,1191]" captionStart="Table 1" captionStartId="1.[818,868,150,166]" captionTargetPageId="1" captionText="Table 1 Kinetic parameters of P. crispum SDH. Vmax - maximal reaction rate of enzyme reaction, K - Michaelis constant of the specific substrate, K -apparent m m Michaelis constant at a given concentration of the second substrate), KSS - inhibition constant at an excess substrate concentration, KA - dissociation constant of the enzyme substrate complex, ♣ for technical reasons (absorbance above 3 or excess substrate inhibition), this parameter could not be determined. Kinetic constants were calculated as an average of values from at least 3 experiments performed in doublets, S. D. are shown." httpUri="http://table.plazi.org/id/DDEF66031C4EFFC2FC9FFF7477A5937F" pageId="1" pageNumber="2" tableUuid="DDEF66031C4EFFC2FC9FFF7477A5937F">Table 1</tableCitation>
).
</paragraph>
<paragraph id="892F368B1C4EFFC2FC9FFB3777879708" blockId="1.[818,1471,1237,1284]" pageId="1" pageNumber="2">
<emphasis id="BBE4EA991C4EFFC2FC9FFB3777879708" bold="true" italics="true" pageId="1" pageNumber="2">2.3. Product inhibition analysis confirmed the ordered mechanism of the SDH-catalyzed reaction in both directions</emphasis>
</paragraph>
<paragraph id="892F368B1C4EFFC2FCFCFACB767E97EF" blockId="1.[818,1488,1321,1982]" pageId="1" pageNumber="2">
The kinetic mechanism of the bisubstrate SDH reaction was analyzed in both directions. To identify the
<typeStatus id="562B88291C4EFFC2FBD0FAA777879754" box="[1149,1190,1349,1368]" pageId="1" pageNumber="2">type</typeStatus>
of mechanism, we constructed Lineweaver-Burk diagnostical plots (
<figureCitation id="11AB2A0E1C4EFFC2FB27FA83779E9778" box="[1162,1215,1377,1396]" captionStart="Fig" captionStartId="2.[100,130,1042,1059]" captionTargetBox="[264,1323,149,1014]" captionTargetId="figure-308@2.[263,1324,148,1015]" captionTargetPageId="2" captionText="Fig. 1. Determination of the type of SDH reaction mechanism based on Lineweaver-Burk diagnostical plots for the direction from SA to DHS (A,B) and from DHS to SA (C,D) Double reciprocal plots are fitted to an equation corresponding to a sequential mechanism." figureDoi="http://doi.org/10.5281/zenodo.8270255" httpUri="https://zenodo.org/record/8270255/files/figure.png" pageId="1" pageNumber="2">Fig. 1</figureCitation>
), a Hanes plot and an Eadie-Hofstee plot (data not shown) and performed product inhibition assays (
<figureCitation id="11AB2A0E1C4EFFC2FC97FA7B704F97A0" box="[826,878,1433,1452]" captionStart="Fig" captionStartId="4.[100,130,148,165]" captionTargetId="figure-7@3.[259,1329,148,1837]" captionTargetPageId="3" captionText="Fig. 2. Product inhibition analysis for the determination of the mechanism of bisubstrate reaction in the direction from SA to DHS (A-F) and in the direction from DHS to SA (G-J). Experimental data are fitted with calculated values determined by non-linear regression using equations charactering competitive and non-competitive inhibition, respectively. SDH products DHS (A,B,C) and NADPH (D,E,F) served as competitive (B,C,D,F) and non-competitive (A,E) inhibitors. The saturating (C,F) and subsaturating (A,B,D,E) concentrations of SA and NADP were 20 mM and 2 mM, and 0.3 mM and 0.5 mM, respectively. Saturating concentrations of NADP and variable concentrations of SA caused no inhibition (N.I.) of NADPH and saturating concentrations of SA and variable concentrations of NADP caused N.I. of DHS (data not shown). SDH products SA (G,H) and NADP (I,J) served as competitive (G,I) and non-competitive (H,J) inhibitors at 0.75 mM (subsaturating concentration) DHS (G,I) and 0.2 mM (subsaturating concentration) NADPH (H,J) as a second substrate. Ki indicates inhibition constants in mM. NC - noncompetitive and C - competitive inhibition. Measurements were performed in doublets (S.D. are shown) and at least 2-4 times (enzyme preparations from different isolations)." figureDoi="http://doi.org/10.5281/zenodo.8270257" httpUri="https://zenodo.org/record/8270257/files/figure.png" pageId="1" pageNumber="2">Fig. 2</figureCitation>
,
<tableCitation id="C41203301C4EFFC2FCD7FA7B70E197A0" box="[890,960,1433,1452]" captionStart="Table 2" captionStartId="2.[100,150,1144,1160]" captionTargetPageId="2" captionText="Table 2 Results of the product inhibition study for the determination of the bisubstrate reaction mechanism of P. crispum SDH. Inhibition constants were calculated as an average of values from 2-4 experiments carried out in doublets, S.D. are shown.NC - non-competitive C - competitive, and N.I. - no inhibition.Patterns and values in brackets mean that the reaction is saturated with the 2nd substrate." httpUri="http://table.plazi.org/id/DDEF66031C4DFFC1FFC9FB9A71BE96D9" pageId="1" pageNumber="2" tableUuid="DDEF66031C4DFFC1FFC9FB9A71BE96D9">Table 2</tableCitation>
). All kinetic parameters including
<subScript id="151434CE1C4EFFC2FAABFA7B761397A2" attach="left" box="[1286,1330,1433,1454]" fontSize="6" pageId="1" pageNumber="2">Vmax</subScript>
,
<subScript id="151434CE1C4EFFC2FA93FA7B767997A2" attach="left" box="[1342,1368,1433,1454]" fontSize="6" pageId="1" pageNumber="2">Km</subScript>
, KA,
<subScript id="151434CE1C4EFFC2FA25FA7B768397A2" attach="left" box="[1416,1442,1433,1454]" fontSize="6" pageId="1" pageNumber="2">Kic</subScript>
,
<subScript id="151434CE1C4EFFC2FA03FA7B76E897A2" attach="left" box="[1454,1481,1433,1454]" fontSize="6" pageId="1" pageNumber="2">Kiu</subScript>
,
<subScript id="151434CE1C4EFFC2FC9FFA57706597C6" attach="left" box="[818,836,1461,1482]" fontSize="6" pageId="1" pageNumber="2">Ki</subScript>
, V*
<subScript id="151434CE1C4EFFC2FCC1FA5E70B197C6" attach="left" box="[876,912,1468,1482]" fontSize="6" pageId="1" pageNumber="2">max,</subScript>
KSS,
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S
<subScript id="151434CE1C4EFFC2FC7CFA5E70C697C6" attach="left" box="[977,999,1468,1482]" fontSize="6" pageId="1" pageNumber="2">0.5</subScript>
</geoCoordinate>
were calculated from non-linear regression using particular equations (
<bibRefCitation id="ED014B7A1C4EFFC2FC50FA32778297EF" author="Bisswanger, H." box="[1021,1187,1488,1507]" pageId="1" pageNumber="2" refId="ref10056" refString="Bisswanger, H., 2002. Enzyme Kinetics. Principles and methods. WILEY-VCH Verlag GmbH, Weinheim." type="book" year="2002">Bisswanger, 2002</bibRefCitation>
;
<bibRefCitation id="ED014B7A1C4EFFC2FB02FA32767097EF" author="Marangoni, A. G." box="[1199,1361,1488,1507]" pageId="1" pageNumber="2" refId="ref12469" refString="Marangoni, A. G., 2003. Enzyme kinetics. A modern approach. John Wiley &amp; Sons, Ltd, Chichester." type="book" year="2003">Marangoni, 2003</bibRefCitation>
).
</paragraph>
<paragraph id="892F368B1C4EFFC2FCFCFA0E778994D3" blockId="1.[818,1488,1321,1982]" pageId="1" pageNumber="2">
The initial rate of the reaction in the physiological direction was measured using several concentrations of DHS and NADPH, showing the typical Lineweaver-Burk plot of a sequential mechanism: straight lines with an intercept left to the ordinate (
<figureCitation id="11AB2A0E1C4EFFC2FB3FF9A277E7945F" box="[1170,1222,1600,1619]" captionStart="Fig" captionStartId="2.[100,130,1042,1059]" captionTargetBox="[264,1323,149,1014]" captionTargetId="figure-308@2.[263,1324,148,1015]" captionTargetPageId="2" captionText="Fig. 1. Determination of the type of SDH reaction mechanism based on Lineweaver-Burk diagnostical plots for the direction from SA to DHS (A,B) and from DHS to SA (C,D) Double reciprocal plots are fitted to an equation corresponding to a sequential mechanism." figureDoi="http://doi.org/10.5281/zenodo.8270255" httpUri="https://zenodo.org/record/8270255/files/figure.png" pageId="1" pageNumber="2">Fig. 1</figureCitation>
) and KA 0.25 ± 0.13 mM. In this direction, the free enzyme binds to NADPH, which allows DHS, but not SA, binding in an ordered mechanism, thus partly explaining the mutual competition between NADPH and SA. NADPH binding apparently prevents NADP binding, and
<emphasis id="BBE4EA991C4EFFC2FBD8F94D77EA94CF" bold="true" box="[1141,1227,1711,1731]" italics="true" pageId="1" pageNumber="2">vice versa</emphasis>
, leading to bilateral mutual competitive inhibition (
<figureCitation id="11AB2A0E1C4EFFC2FBBFF929776694D3" box="[1042,1095,1739,1759]" captionStart="Fig" captionStartId="4.[100,130,1529,1546]" captionTargetBox="[345,1246,439,1500]" captionTargetId="figure-625@4.[339,1249,437,1501]" captionTargetPageId="4" captionText="Fig. 3. Proposed kinetic mechanism of the bisubstrate reaction catalyzed by P. crispum SDH from non-photosynthetic tissue. SDH reaction scheme (A), graphical representation of the product inhibition study in DHS reduction direction (B) and in SA oxidation direction (C) with a formation of dead-end complexes. Non-Competitive (NC) and competitive (C) product inhibitors are indicated." figureDoi="http://doi.org/10.5281/zenodo.8270259" httpUri="https://zenodo.org/record/8270259/files/figure.png" pageId="1" pageNumber="2">Fig. 3</figureCitation>
,
<tableCitation id="C41203301C4EFFC2FBF9F92977BB94D3" box="[1108,1178,1739,1759]" captionStart="Table 2" captionStartId="2.[100,150,1144,1160]" captionTargetPageId="2" captionText="Table 2 Results of the product inhibition study for the determination of the bisubstrate reaction mechanism of P. crispum SDH. Inhibition constants were calculated as an average of values from 2-4 experiments carried out in doublets, S.D. are shown.NC - non-competitive C - competitive, and N.I. - no inhibition.Patterns and values in brackets mean that the reaction is saturated with the 2nd substrate." httpUri="http://table.plazi.org/id/DDEF66031C4DFFC1FFC9FB9A71BE96D9" pageId="1" pageNumber="2" tableUuid="DDEF66031C4DFFC1FFC9FB9A71BE96D9">Table 2</tableCitation>
).
</paragraph>
<paragraph id="892F368B1C4EFFC2FCFCF905705C958A" blockId="1.[818,1488,1321,1982]" pageId="1" pageNumber="2">
The direction of SA oxidation confirmed the strong affinity between the free enzyme and NADP. The mechanism was sequential but with a very low, almost immeasurable, dissociation constant for the complex enzyme substrate (KA), thus it seems to look like a ping-pong. Obviously, the complex enzyme-NADP is very thermodynamically stable (
<figureCitation id="11AB2A0E1C4EFFC2FC97F891704E958A" box="[826,879,1907,1926]" captionStart="Fig" captionStartId="2.[100,130,1042,1059]" captionTargetBox="[264,1323,149,1014]" captionTargetId="figure-308@2.[263,1324,148,1015]" captionTargetPageId="2" captionText="Fig. 1. Determination of the type of SDH reaction mechanism based on Lineweaver-Burk diagnostical plots for the direction from SA to DHS (A,B) and from DHS to SA (C,D) Double reciprocal plots are fitted to an equation corresponding to a sequential mechanism." figureDoi="http://doi.org/10.5281/zenodo.8270255" httpUri="https://zenodo.org/record/8270255/files/figure.png" pageId="1" pageNumber="2">Fig. 1</figureCitation>
).
</paragraph>
<paragraph id="892F368B1C4EFFC1FCFCF86D712E947C" blockId="1.[818,1488,1321,1982]" lastBlockId="2.[100,770,1545,1955]" lastPageId="2" lastPageNumber="3" pageId="1" pageNumber="2">
NADP binding enables both DHS and SA binding. The latter follows a classical ordered mechanism in the direction of SA + NADP → NADPH + DHS; in contrast, the former results in the formation of a dead-end complex. Its origin explains the competitive inhibition of DHS against the substrate SA. Thus, these findings further confirm the competition between NADP and NADPH (
<figureCitation id="11AB2A0E1C4DFFC1FED4F9BF728F947C" box="[377,430,1629,1648]" captionStart="Fig" captionStartId="4.[100,130,1529,1546]" captionTargetBox="[345,1246,439,1500]" captionTargetId="figure-625@4.[339,1249,437,1501]" captionTargetPageId="4" captionText="Fig. 3. Proposed kinetic mechanism of the bisubstrate reaction catalyzed by P. crispum SDH from non-photosynthetic tissue. SDH reaction scheme (A), graphical representation of the product inhibition study in DHS reduction direction (B) and in SA oxidation direction (C) with a formation of dead-end complexes. Non-Competitive (NC) and competitive (C) product inhibitors are indicated." figureDoi="http://doi.org/10.5281/zenodo.8270259" httpUri="https://zenodo.org/record/8270259/files/figure.png" pageId="2" pageNumber="3">Fig. 3</figureCitation>
,
<tableCitation id="C41203301C4DFFC1FE16F9BF7120947C" box="[443,513,1629,1648]" captionStart="Table 2" captionStartId="2.[100,150,1144,1160]" captionTargetPageId="2" captionText="Table 2 Results of the product inhibition study for the determination of the bisubstrate reaction mechanism of P. crispum SDH. Inhibition constants were calculated as an average of values from 2-4 experiments carried out in doublets, S.D. are shown.NC - non-competitive C - competitive, and N.I. - no inhibition.Patterns and values in brackets mean that the reaction is saturated with the 2nd substrate." httpUri="http://table.plazi.org/id/DDEF66031C4DFFC1FFC9FB9A71BE96D9" pageId="2" pageNumber="3" tableUuid="DDEF66031C4DFFC1FFC9FB9A71BE96D9">Table 2</tableCitation>
).
</paragraph>
<caption id="DDEF66031C4DFFC1FFC9FBF0771C9631" ID-DOI="http://doi.org/10.5281/zenodo.8270255" ID-Zenodo-Dep="8270255" httpUri="https://zenodo.org/record/8270255/files/figure.png" pageId="2" pageNumber="3" startId="2.[100,130,1042,1059]" targetBox="[264,1323,149,1014]" targetPageId="2" targetType="figure">
<paragraph id="892F368B1C4DFFC1FFC9FBF0771C9631" blockId="2.[100,1487,1042,1085]" pageId="2" pageNumber="3">
<emphasis id="BBE4EA991C4DFFC1FFC9FBF072089631" bold="true" pageId="2" pageNumber="3">Fig. 1. Determination of the type of SDH reaction mechanism based on Lineweaver-Burk diagnostical plots for the direction from SA to DHS (A,B) and from DHS to SA (C,D)</emphasis>
Double reciprocal plots are fitted to an equation corresponding to a sequential mechanism.
</paragraph>
</caption>
<caption id="DDEF66031C4DFFC1FFC9FB9A71BE96D9" ID-Table-UUID="DDEF66031C4DFFC1FFC9FB9A71BE96D9" httpUri="http://table.plazi.org/id/DDEF66031C4DFFC1FFC9FB9A71BE96D9" pageId="2" pageNumber="3" startId="2.[100,150,1144,1160]" targetBox="[116,1471,1257,1491]" targetIsTable="true" targetPageId="2" targetType="table">
<paragraph id="892F368B1C4DFFC1FFC9FB9A73869685" blockId="2.[100,1487,1144,1238]" box="[100,167,1144,1161]" pageId="2" pageNumber="3">
<emphasis id="BBE4EA991C4DFFC1FFC9FB9A73869685" bold="true" box="[100,167,1144,1161]" pageId="2" pageNumber="3">Table 2</emphasis>
</paragraph>
<paragraph id="892F368B1C4DFFC1FFC9FB7071BE96D9" blockId="2.[100,1487,1144,1238]" pageId="2" pageNumber="3">
<emphasis id="BBE4EA991C4DFFC1FFC9FB7070D696AE" bold="true" box="[100,1015,1169,1186]" pageId="2" pageNumber="3">Results of the product inhibition study for the determination of the bisubstrate reaction mechanism of</emphasis>
<taxonomicName id="4E904D081C4DFFC1FC56FB7377FE96AE" authority="SDH. Inhibition" authorityName="SDH. Inhibition" box="[1019,1247,1169,1187]" class="Magnoliopsida" family="Apiaceae" genus="Petroselinum" kingdom="Plantae" order="Apiales" pageId="2" pageNumber="3" phylum="Tracheophyta" rank="species" species="crispum">
<emphasis id="BBE4EA991C4DFFC1FC56FB7377A696AE" bold="true" box="[1019,1159,1169,1187]" pageId="2" pageNumber="3">
<emphasis id="BBE4EA991C4DFFC1FC56FB73777496AF" bold="true" box="[1019,1109,1169,1187]" italics="true" pageId="2" pageNumber="3">P. crispum</emphasis>
SDH.
</emphasis>
Inhibition
</taxonomicName>
constants were calculated as an average of values from 2-4 experiments carried out in doublets, S.D. are shown.NC - non-competitive C - competitive, and N.I. - no inhibition. Patterns and values in brackets mean that the reaction is saturated with the 2
<superScript id="7EE59BC31C4DFFC1FD9BFB22716796C0" attach="left" box="[566,582,1216,1228]" fontSize="5" pageId="2" pageNumber="3">nd</superScript>
substrate.
</paragraph>
</caption>
<paragraph id="892F368B1C4DFFC1FEA0FB0B70C497DF" pageId="2" pageNumber="3">
<table id="FB90C42B1C4D003CFFD9FB0B769E97DF" box="[116,1471,1257,1491]" gridcols="9" gridrows="9" pageId="2" pageNumber="3">
<tr id="37A034C91C4D003CFFD9FB0B769E96FB" box="[116,1471,1257,1271]" gridrow="0" pageId="2" pageNumber="3" rowspan-0="1" rowspan-3="1" rowspan-4="1" rowspan-6="1" rowspan-7="1" rowspan-8="1">
<th id="74715DB51C4D003CFEA0FB0B72C596FB" box="[269,484,1257,1271]" colspan="2" colspanRight="1" gridcol="1" gridrow="0" pageId="2" pageNumber="3">Product inhibitor</th>
<th id="74715DB51C4D003CFCA3FB0B70C496FB" box="[782,997,1257,1271]" gridcol="5" gridrow="0" pageId="2" pageNumber="3">Product inhibitor</th>
</tr>
<tr id="37A034C91C4D003CFFD9FAEE769E9717" box="[116,1471,1292,1307]" gridrow="1" pageId="2" pageNumber="3" rowspan-0="1" rowspan-2="1" rowspan-4="1" rowspan-6="1" rowspan-8="1">
<td id="74715DB51C4D003CFEA0FAEE72A99717" box="[269,392,1292,1307]" gridcol="1" gridrow="1" pageId="2" pageNumber="3">Q (NADP)</td>
<td id="74715DB51C4D003CFDB8FAEE71409717" box="[533,609,1292,1307]" gridcol="3" gridrow="1" pageId="2" pageNumber="3">P (SA)</td>
<td id="74715DB51C4D003CFCA3FAEE70C49717" box="[782,997,1292,1307]" gridcol="5" gridrow="1" pageId="2" pageNumber="3">A (NADPH)</td>
<td id="74715DB51C4D003CFBD3FAEE77E79717" box="[1150,1222,1292,1307]" gridcol="7" gridrow="1" pageId="2" pageNumber="3">B (DHS)</td>
</tr>
<tr id="37A034C91C4D003CFFD9FAC1769E973D" box="[116,1471,1315,1329]" gridrow="2" pageId="2" pageNumber="3" rowspan-0="1" rowspan-2="1" rowspan-3="1" rowspan-4="1" rowspan-6="1" rowspan-7="1" rowspan-8="1">
<td id="74715DB51C4D003CFEA0FAC172A9973D" box="[269,392,1315,1329]" gridcol="1" gridrow="2" pageId="2" pageNumber="3">Varied substrate</td>
<td id="74715DB51C4D003CFCA3FAC170C4973D" box="[782,997,1315,1329]" gridcol="5" gridrow="2" pageId="2" pageNumber="3">Varied substrate</td>
</tr>
<tr id="37A034C91C4D003CFFD9FAD8769E9744" box="[116,1471,1338,1352]" gridrow="3" pageId="2" pageNumber="3">
<th id="74715DB51C4D003CFFD9FAD873FB9744" box="[116,218,1338,1352]" gridcol="0" gridrow="3" pageId="2" pageNumber="3">i.e.</th>
<td id="74715DB51C4D003CFEA0FAD872A99744" box="[269,392,1338,1352]" gridcol="1" gridrow="3" pageId="2" pageNumber="3">1/NADPH</td>
<td id="74715DB51C4D003CFE37FAD872C59744" box="[410,484,1338,1352]" gridcol="2" gridrow="3" pageId="2" pageNumber="3">1/DHS</td>
<td id="74715DB51C4D003CFDB8FAD871409744" box="[533,609,1338,1352]" gridcol="3" gridrow="3" pageId="2" pageNumber="3">1/NADPH</td>
<td id="74715DB51C4D003CFD39FAD871FA9744" box="[660,731,1338,1352]" gridcol="4" gridrow="3" pageId="2" pageNumber="3">1/DHS</td>
<td id="74715DB51C4D003CFCA3FAD870C49744" box="[782,997,1338,1352]" gridcol="5" gridrow="3" pageId="2" pageNumber="3">1/NADP</td>
<td id="74715DB51C4D003CFBAAFAD8776D9744" box="[1031,1100,1338,1352]" gridcol="6" gridrow="3" pageId="2" pageNumber="3">1/SA</td>
<td id="74715DB51C4D003CFBD3FAD877E79744" box="[1150,1222,1338,1352]" gridcol="7" gridrow="3" pageId="2" pageNumber="3">1/NADP</td>
<td id="74715DB51C4D003CFB54FAD8769E9744" box="[1273,1471,1338,1352]" gridcol="8" gridrow="3" pageId="2" pageNumber="3">1/SA</td>
</tr>
<tr id="37A034C91C4D003CFFD9FABF769E9767" box="[116,1471,1373,1387]" gridrow="4" pageId="2" pageNumber="3" rowspan-2="1" rowspan-3="1" rowspan-4="1" rowspan-6="1" rowspan-7="1" rowspan-8="1">
<th id="74715DB51C4D003CFFD9FABF73FB9767" box="[116,218,1373,1387]" gridcol="0" gridrow="4" pageId="2" pageNumber="3">2nd substrate</th>
<td id="74715DB51C4D003CFEA0FABF72A99767" box="[269,392,1373,1387]" gridcol="1" gridrow="4" pageId="2" pageNumber="3">subsaturated</td>
<td id="74715DB51C4D003CFCA3FABF70C49767" box="[782,997,1373,1387]" gridcol="5" gridrow="4" pageId="2" pageNumber="3">subsaturated (saturated)</td>
</tr>
<tr id="37A034C91C4D003CFFD9FA62769E9783" box="[116,1471,1408,1423]" gridrow="5" pageId="2" pageNumber="3">
<th id="74715DB51C4D003CFFD9FA6273FB9783" box="[116,218,1408,1423]" gridcol="0" gridrow="5" pageId="2" pageNumber="3">Pattern</th>
<td id="74715DB51C4D003CFEA0FA6272A99783" box="[269,392,1408,1423]" gridcol="1" gridrow="5" pageId="2" pageNumber="3">C</td>
<td id="74715DB51C4D003CFE37FA6272C59783" box="[410,484,1408,1423]" gridcol="2" gridrow="5" pageId="2" pageNumber="3">NC</td>
<td id="74715DB51C4D003CFDB8FA6271409783" box="[533,609,1408,1423]" gridcol="3" gridrow="5" pageId="2" pageNumber="3">C</td>
<td id="74715DB51C4D003CFD39FA6271FA9783" box="[660,731,1408,1423]" gridcol="4" gridrow="5" pageId="2" pageNumber="3">NC</td>
<td id="74715DB51C4D003CFCA3FA6270C49783" box="[782,997,1408,1423]" gridcol="5" gridrow="5" pageId="2" pageNumber="3">C (C)</td>
<td id="74715DB51C4D003CFBAAFA62776D9783" box="[1031,1100,1408,1423]" gridcol="6" gridrow="5" pageId="2" pageNumber="3">NC (N.I.)</td>
<td id="74715DB51C4D003CFBD3FA6277E79783" box="[1150,1222,1408,1423]" gridcol="7" gridrow="5" pageId="2" pageNumber="3">NC (N.I.)</td>
<td id="74715DB51C4D003CFB54FA62769E9783" box="[1273,1471,1408,1423]" gridcol="8" gridrow="5" pageId="2" pageNumber="3">C (C)</td>
</tr>
<tr id="37A034C91C4D003CFFD9FA75769E97A9" box="[116,1471,1431,1445]" gridrow="6" pageId="2" pageNumber="3">
<th id="74715DB51C4D003CFFD9FA7573FB97A9" box="[116,218,1431,1445]" gridcol="0" gridrow="6" pageId="2" pageNumber="3">Ki [mM]</th>
<td id="74715DB51C4D003CFEA0FA7572A997A9" box="[269,392,1431,1445]" gridcol="1" gridrow="6" pageId="2" pageNumber="3">0.22 ± 0.02</td>
<td id="74715DB51C4D003CFE37FA7572C597A9" box="[410,484,1431,1445]" gridcol="2" gridrow="6" pageId="2" pageNumber="3">0.4 ± 0.1</td>
<td id="74715DB51C4D003CFDB8FA75714097A9" box="[533,609,1431,1445]" gridcol="3" gridrow="6" pageId="2" pageNumber="3">0.7 ± 0.1</td>
<td id="74715DB51C4D003CFD39FA7571FA97A9" box="[660,731,1431,1445]" gridcol="4" gridrow="6" pageId="2" pageNumber="3">0.7 ± 0.3</td>
<td id="74715DB51C4D003CFCA3FA7570C497A9" box="[782,997,1431,1445]" gridcol="5" gridrow="6" pageId="2" pageNumber="3">0.12 ± 0.07 (0.49 ± 0.03)</td>
<td id="74715DB51C4D003CFBAAFA75776D97A9" box="[1031,1100,1431,1445]" gridcol="6" gridrow="6" pageId="2" pageNumber="3">0.36±</td>
<td id="74715DB51C4D003CFBD3FA7577E797A9" box="[1150,1222,1431,1445]" gridcol="7" gridrow="6" pageId="2" pageNumber="3">1.4 ± 0.2</td>
<td id="74715DB51C4D003CFB54FA75769E97A9" box="[1273,1471,1431,1445]" gridcol="8" gridrow="6" pageId="2" pageNumber="3">0.13 ± 0.03 (0.33 ± 0.03)</td>
</tr>
<tr id="37A034C91C4D003CFFD9FA4C769E97B0" box="[116,1471,1454,1468]" gridrow="7" pageId="2" pageNumber="3" rowspan-0="1" rowspan-1="1" rowspan-2="1" rowspan-3="1" rowspan-4="1" rowspan-5="1" rowspan-7="1" rowspan-8="1">
<td id="74715DB51C4D003CFBAAFA4C776D97B0" box="[1031,1100,1454,1468]" gridcol="6" gridrow="7" pageId="2" pageNumber="3">0.08</td>
</tr>
<tr id="37A034C91C4D003CFFD9FA27769E97DF" box="[116,1471,1477,1491]" gridrow="8" pageId="2" pageNumber="3" rowspan-3="1" rowspan-4="1" rowspan-6="1" rowspan-7="1" rowspan-8="1">
<th id="74715DB51C4D003CFFD9FA2773FB97DF" box="[116,218,1477,1491]" gridcol="0" gridrow="8" pageId="2" pageNumber="3">Mechanism</th>
<td id="74715DB51C4D003CFEA0FA2772C597DF" box="[269,484,1477,1491]" colspan="2" colspanRight="1" gridcol="1" gridrow="8" pageId="2" pageNumber="3">steady state sequential order</td>
<td id="74715DB51C4D003CFCA3FA2770C497DF" box="[782,997,1477,1491]" gridcol="5" gridrow="8" pageId="2" pageNumber="3">steady state sequential order</td>
</tr>
</table>
</paragraph>
<paragraph id="892F368B1C4DFFC1FF29F99B72C594F7" blockId="2.[100,770,1545,1955]" pageId="2" pageNumber="3">
DHS binding to the free enzyme prevents NADPH binding by forming a dead-end complex (enzyme-DHS) that precludes a disordered mechanism in the direction of NADPH + DHS → SA + NADP. In addition, another dead-end complex (enzyme-DHS-NADP) is also formed when NADP binds to the enzyme-DHS (
<figureCitation id="11AB2A0E1C4DFFC1FE0CF90A72F794F7" box="[417,470,1768,1787]" captionStart="Fig" captionStartId="4.[100,130,1529,1546]" captionTargetBox="[345,1246,439,1500]" captionTargetId="figure-625@4.[339,1249,437,1501]" captionTargetPageId="4" captionText="Fig. 3. Proposed kinetic mechanism of the bisubstrate reaction catalyzed by P. crispum SDH from non-photosynthetic tissue. SDH reaction scheme (A), graphical representation of the product inhibition study in DHS reduction direction (B) and in SA oxidation direction (C) with a formation of dead-end complexes. Non-Competitive (NC) and competitive (C) product inhibitors are indicated." figureDoi="http://doi.org/10.5281/zenodo.8270259" httpUri="https://zenodo.org/record/8270259/files/figure.png" pageId="2" pageNumber="3">Fig. 3</figureCitation>
).
</paragraph>
<paragraph id="892F368B1C4DFFC1FF29F8E670D594A4" blockId="2.[100,770,1545,1955]" lastBlockId="2.[818,1488,1545,1704]" pageId="2" pageNumber="3">
SA binding to the free enzyme prevents both NADP and NADPH binding also by forming a dead-end complex, which, in the case of NADP, prevents a disordered mechanism in the direction of SA + NADP → NADPH + DHS. In the case of NADPH, the formation of the dead-end complex completes the explanation for the competitive inhibition by the SA inhibitor against the NADPH substrate. Thus, SA competes with NADPH to bind to free enzyme, but not the other way around, that is, NADPH does not compete with SA since SA binding to the enzyme leads to a dead-end complex. Therefore, we proposed an ordered mechanism of the bi-substrate reaction catalyzed by SDH in both directions with three dead-end complexes (enzyme-DHS, enzyme-SA, and enzymeNADP-DHS) (
<figureCitation id="11AB2A0E1C4DFFC1FC1CF97770C694A4" box="[945,999,1685,1704]" captionStart="Fig" captionStartId="4.[100,130,1529,1546]" captionTargetBox="[345,1246,439,1500]" captionTargetId="figure-625@4.[339,1249,437,1501]" captionTargetPageId="4" captionText="Fig. 3. Proposed kinetic mechanism of the bisubstrate reaction catalyzed by P. crispum SDH from non-photosynthetic tissue. SDH reaction scheme (A), graphical representation of the product inhibition study in DHS reduction direction (B) and in SA oxidation direction (C) with a formation of dead-end complexes. Non-Competitive (NC) and competitive (C) product inhibitors are indicated." figureDoi="http://doi.org/10.5281/zenodo.8270259" httpUri="https://zenodo.org/record/8270259/files/figure.png" pageId="2" pageNumber="3">Fig. 3</figureCitation>
).
</paragraph>
</subSubSection>
</treatment>
</document>