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An Investigation of Peroxidase Activity in Biomimetic and Biological Systems

Ryabova, Ekaterina LU (2004)
Abstract (Swedish)
Popular Abstract in Swedish

Järn är den mest vanligt förekommande övergångsmetallen i alla levande organismer, från bakterier till växter och djur och även i människan. Järn är en viktig del av olika proteiner och förekommer mycket ofta i form av en hemgrupp (se bild). Enzymer är proteiner som kan katalysera biokemiska processer. Det ställe i enzymet, där reaktionen sker, kallas enzymets aktiva säte. Det aktiva sätet kan innehålla en kofaktor (t.ex. hem) som är omgiven av proteinets aminosyror. En av de viktigaste klasserna av proteiner och enzymer kallas hemproteiner. Dessa har hem som kofaktor. Prefixet hem (eller hemo-) hänvisar till järn(II)porfyrin, medan hemin anger järn(III)porfyrin. Det mest kända hemproteinet är... (More)
Popular Abstract in Swedish

Järn är den mest vanligt förekommande övergångsmetallen i alla levande organismer, från bakterier till växter och djur och även i människan. Järn är en viktig del av olika proteiner och förekommer mycket ofta i form av en hemgrupp (se bild). Enzymer är proteiner som kan katalysera biokemiska processer. Det ställe i enzymet, där reaktionen sker, kallas enzymets aktiva säte. Det aktiva sätet kan innehålla en kofaktor (t.ex. hem) som är omgiven av proteinets aminosyror. En av de viktigaste klasserna av proteiner och enzymer kallas hemproteiner. Dessa har hem som kofaktor. Prefixet hem (eller hemo-) hänvisar till järn(II)porfyrin, medan hemin anger järn(III)porfyrin. Det mest kända hemproteinet är hemoglobin som utför transport av syre från lungorna till kroppens alla organ. Peroxidaser och katalaser tillhör den största gruppen av heminnehållande enzymer som ofta kallas ”skyddsenzymer”. Dessa enzymer skyddar levande organismer mot okontrollerad oxidation med väteperoxid. Peroxidaser använder väteperoxid för oxidation av en stor mängd organiska och oorganiska molekyler. Pepparrotsperoxidas är det mest studerade peroxidasenzymet. Det har stor användning inom medicin, cellbiologi, vid miljökontroll samt inom livsmedelsindustrin. Modellföreningar används ofta i enzymrelaterade studier. I praktiken är det ofta lättare att studera små modellmolekyler med olika experimentella metoder och med högre noggrannhet än de naturliga enzymerna. Modellföreningar med låg molekylvikt och bra peroxidasaktivitet kan, för vissa praktiska tillämpningar, vara ett bättre alternativ till naturliga enzymer. I denna avhandling har peroxidasaktivitet för flera hemin-peptid-komplex undersökts. De första modellkomplexen baserades på hemin, som ingår i det aktiva sätet hos de flesta hemoglobiner och peroxidaser. Peptidkedjor av olika längd kopplades till hemins propionater (positionerna 6 och 7) och komplex med peptider kopplade till såväl en som två propionater framställdes. Den sista aminosyran i peptiden var alltid histidin, eftersom dess imidazolinnehållande sidokedja binder starkt till järnatomen i hemin. Denna bindning anses höja reaktiviteten hos hemin-peptidkomplexen vid deras reaktion med väteperoxid. Reaktivitetsstudier med dessa modellkomplex visar att både mono- och de bis-substituerade hemin-peptid komplexen har katalytisk aktivitet. Komplex med längre peptidkedjor uppvisade högre aktivitet. Detta kan sannolikt bero på den struktur som erhålles med en längre peptidkedja förväntas vara mindre spänd. Modeller baserade på mesohemin och deuterohemin visade högre reaktivitet än heminbaserade modeller, men dessa peptidsubstituerade modeller visade inte högre reaktivitet än de ursprungliga, icke substituerade, hemin, meso- och deuterohemin, möjligtvis på grund av svag histidin-järnbindning. Modifikationen med peptider har dock gjort heminkomplexen lösliga i vatten och vanliga organiska lösningsmedel, vilket inte är fallet med osubstituerad hemin. Vid högre koncentration av väteperoxid har modellerna dessutom visat högre aktivitet jämfört med icke modifierat hemin. Den katalytiska aktiviteten i modellkomplex med mangan(III) i stället för järn(III) var mycket bättre och kan jämföras med aktiviteten hos de mini-enzymer som kallas mikroperoxidaser. Detta kan förklaras med större stabilitet för själva mangankomplexet och för de intermediater som bildas vid reaktion med väteperoxid. Vitreoscilla hemoglobin (VHb) upptäcktes först i Vitreoscilla stercoraria-bakterien. Det har två lika delar som båda har en hem i sin aktiva säte. Möjliga funktioner för Vhb diskuteras fortfarande, men det har föreslagits att detta hemoglobin kan ha fler funktioner än bara transport av syre. Resultaten i denna avhandling visar att Vitreoscilla hemoglobin har en peroxidasaktivitet som är jämförbar med den för pepparrotsperoxidas. (Less)
Abstract
In order to create a heme environment which permits biomimicry of heme-containing plant peroxidases and investigate the role played by the vinyl side chains of the porphyrin ring on the stability and, possibly, catalytic activity of model compounds, a number of new synthetic microperoxidases -– hemin-6(7)-gly-his methyl ester (HGH) and hemin-6(7)-gly-gly-his methyl ester (HGGH) and hemin-6,7-bis(gly-gly-his methyl ester) (H2GGH) as well as mesohemin-6(7)-gly-gly-his methyl ester (MGGH) and deuterohemin-6(7)-gly-gly-his methyl ester (DGGH) have been prepared by condensation of a peptide residue with the propionic side chains of hemin, mesohemin and deuterohemin, respectively. Reactivity studies towards different organic and inorganic... (More)
In order to create a heme environment which permits biomimicry of heme-containing plant peroxidases and investigate the role played by the vinyl side chains of the porphyrin ring on the stability and, possibly, catalytic activity of model compounds, a number of new synthetic microperoxidases -– hemin-6(7)-gly-his methyl ester (HGH) and hemin-6(7)-gly-gly-his methyl ester (HGGH) and hemin-6,7-bis(gly-gly-his methyl ester) (H2GGH) as well as mesohemin-6(7)-gly-gly-his methyl ester (MGGH) and deuterohemin-6(7)-gly-gly-his methyl ester (DGGH) have been prepared by condensation of a peptide residue with the propionic side chains of hemin, mesohemin and deuterohemin, respectively. Reactivity studies towards different organic and inorganic substrates showed that not only five- but also six-coordinate hemin peptide complexes catalyze the oxidation of substrates by H2O2. The HGGH complex showed higher catalytic activity than HGH due to a less strained structure provided by the longer peptide arm. Microperoxidases based on meso- and deuterohemin (MGGH and DGGH, respectively) exhibited at least one order of magnitude higher reactivity relative to their hemin-based analogues. However, computational modelling showed that the relative energy differences between the different hemins are small, suggesting that more subtle factors (e.g. properties of reaction media) rather that only electronic properties of substituents of the porphyrin ring determine the reactivity order of the hemin peptide complexes studied. In general, the hemin-peptide complexes showed relatively low peroxidase reactivity. On the contrary, manganese(III) microperoxidase (MnGGH) showed relatively high reactivity in peroxidase-type reactions. Rate constants for Compound I formation obtained by fast kinetics studies as well as steady-state rate constants for the reaction of MnGGH with the substrates were comparable to these observed for manganese microperoxidase-8 (MnMP-8). The higher peroxidase reactivity of MnGGH as compared to its iron(III) analogue, HGGH, may be attributed to the higher operational stability of the manganese complexes as well as higher stability of Mn(III) oxo intermediates. An investigation of the peroxidase reactivity of bacterial hemoglobin from Vitreoscilla stercoraria (VHb) showed for the first time that VHb does exhibit peroxidase catalytic activity which for certain substrates is comparable with the activity of horseradish peroxidase. VHb also showed unusual substrate specificity (for peroxidases); i.e. good activity was observed only for substrates with more than one hydrogen bond donor/acceptor. In order to create a heme environment which permits biomimicry of heme-containing plant peroxidases and investigate the role played by the vinyl side chains of the porphyrin ring on the stability and catalytic activity of model compounds, a number of new synthetic microperoxidases – hemin-6(7)-gly-his methyl ester (HGH), hemin-6(7)-gly-gly-his methyl ester (HGGH) and hemin-6,7-bis(gly-gly-his methyl ester) (H2GGH) as well as mesohemin-6(7)-gly-gly-his methyl ester (MGGH) and deuterohemin-6(7)-gly-gly-his methyl ester (DGGH) have been prepared by condensation of a peptide residue with the propionic acid side chains of hemin, mesohemin and deuterohemin, respectively. Reactivity studies towards different organic and inorganic substrates showed that not only five- but also six-coordinate hemin peptide complexes catalyze the oxidation of substrates by H2O2. The HGGH complex showed higher catalytic activity than HGH, possibly due to a less strained structure provided by the longer peptide arm. Microperoxidases based on meso- and deuterohemin (MGGH and DGGH, respectively) exhibited at least one order of magnitude higher reactivity relative to their hemin-based analogues. However, computational modelling showed that the relative energy differences between the different hemins are small, suggesting that more subtle factors (e.g. properties of reaction media) rather that only electronic properties of the substituents of the porphyrin ring determine the reactivity order of the hemin peptide complexes studied. Manganese(III) microperoxidase (MnGGH) showed relatively high reactivity in peroxidase-type reactions. Rate constants for Compound I formation obtained by fast kinetics studies as well as steady-state rate constants for the reaction of MnGGH with substrates were comparable to these observed for manganese microperoxidase-8. The higher peroxidase reactivity of MnGGH as compared to its iron(III) analogue, HGGH, may be attributed to the higher operational stability of the manganese complex as well as higher stability of Mn(III) oxo intermediates. An investigation of the peroxidase reactivity of the bacterial hemoglobin from Vitreoscilla stercoraria (VHb) showed for the first time that VHb does exhibit peroxidase catalytic activity which for certain substrates is comparable with the activity of horseradish peroxidase. VHb also showed unusual substrate specificity (for peroxidases); i.e. good activity was observed only for substrates with more than one hydrogen bond donor/acceptor. (Less)
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author
opponent
  • Prof Casella, Luigi
organization
publishing date
type
Thesis
publication status
published
subject
keywords
metabolism, Metabolism, enzymologi, Proteiner, enzymology, Proteins, hemoglobin, catalysis, peptide, peroxidase, protoporphyrin IX, Biokemi, Biochemistry
pages
161 pages
publisher
Inorganic Chemistry, Chemical Center, Lund University
defense location
sal F Kemicentrum
defense date
2004-12-17 10:30
ISBN
91-7422-065-9
language
English
LU publication?
yes
id
e489b287-d5e2-4ec3-9ab6-139a2c3bbacc (old id 467615)
date added to LUP
2007-10-13 15:05:42
date last changed
2016-09-19 08:45:05
@misc{e489b287-d5e2-4ec3-9ab6-139a2c3bbacc,
  abstract     = {In order to create a heme environment which permits biomimicry of heme-containing plant peroxidases and investigate the role played by the vinyl side chains of the porphyrin ring on the stability and, possibly, catalytic activity of model compounds, a number of new synthetic microperoxidases -– hemin-6(7)-gly-his methyl ester (HGH) and hemin-6(7)-gly-gly-his methyl ester (HGGH) and hemin-6,7-bis(gly-gly-his methyl ester) (H2GGH) as well as mesohemin-6(7)-gly-gly-his methyl ester (MGGH) and deuterohemin-6(7)-gly-gly-his methyl ester (DGGH) have been prepared by condensation of a peptide residue with the propionic side chains of hemin, mesohemin and deuterohemin, respectively. Reactivity studies towards different organic and inorganic substrates showed that not only five- but also six-coordinate hemin peptide complexes catalyze the oxidation of substrates by H2O2. The HGGH complex showed higher catalytic activity than HGH due to a less strained structure provided by the longer peptide arm. Microperoxidases based on meso- and deuterohemin (MGGH and DGGH, respectively) exhibited at least one order of magnitude higher reactivity relative to their hemin-based analogues. However, computational modelling showed that the relative energy differences between the different hemins are small, suggesting that more subtle factors (e.g. properties of reaction media) rather that only electronic properties of substituents of the porphyrin ring determine the reactivity order of the hemin peptide complexes studied. In general, the hemin-peptide complexes showed relatively low peroxidase reactivity. On the contrary, manganese(III) microperoxidase (MnGGH) showed relatively high reactivity in peroxidase-type reactions. Rate constants for Compound I formation obtained by fast kinetics studies as well as steady-state rate constants for the reaction of MnGGH with the substrates were comparable to these observed for manganese microperoxidase-8 (MnMP-8). The higher peroxidase reactivity of MnGGH as compared to its iron(III) analogue, HGGH, may be attributed to the higher operational stability of the manganese complexes as well as higher stability of Mn(III) oxo intermediates. An investigation of the peroxidase reactivity of bacterial hemoglobin from Vitreoscilla stercoraria (VHb) showed for the first time that VHb does exhibit peroxidase catalytic activity which for certain substrates is comparable with the activity of horseradish peroxidase. VHb also showed unusual substrate specificity (for peroxidases); i.e. good activity was observed only for substrates with more than one hydrogen bond donor/acceptor. In order to create a heme environment which permits biomimicry of heme-containing plant peroxidases and investigate the role played by the vinyl side chains of the porphyrin ring on the stability and catalytic activity of model compounds, a number of new synthetic microperoxidases – hemin-6(7)-gly-his methyl ester (HGH), hemin-6(7)-gly-gly-his methyl ester (HGGH) and hemin-6,7-bis(gly-gly-his methyl ester) (H2GGH) as well as mesohemin-6(7)-gly-gly-his methyl ester (MGGH) and deuterohemin-6(7)-gly-gly-his methyl ester (DGGH) have been prepared by condensation of a peptide residue with the propionic acid side chains of hemin, mesohemin and deuterohemin, respectively. Reactivity studies towards different organic and inorganic substrates showed that not only five- but also six-coordinate hemin peptide complexes catalyze the oxidation of substrates by H2O2. The HGGH complex showed higher catalytic activity than HGH, possibly due to a less strained structure provided by the longer peptide arm. Microperoxidases based on meso- and deuterohemin (MGGH and DGGH, respectively) exhibited at least one order of magnitude higher reactivity relative to their hemin-based analogues. However, computational modelling showed that the relative energy differences between the different hemins are small, suggesting that more subtle factors (e.g. properties of reaction media) rather that only electronic properties of the substituents of the porphyrin ring determine the reactivity order of the hemin peptide complexes studied. Manganese(III) microperoxidase (MnGGH) showed relatively high reactivity in peroxidase-type reactions. Rate constants for Compound I formation obtained by fast kinetics studies as well as steady-state rate constants for the reaction of MnGGH with substrates were comparable to these observed for manganese microperoxidase-8. The higher peroxidase reactivity of MnGGH as compared to its iron(III) analogue, HGGH, may be attributed to the higher operational stability of the manganese complex as well as higher stability of Mn(III) oxo intermediates. An investigation of the peroxidase reactivity of the bacterial hemoglobin from Vitreoscilla stercoraria (VHb) showed for the first time that VHb does exhibit peroxidase catalytic activity which for certain substrates is comparable with the activity of horseradish peroxidase. VHb also showed unusual substrate specificity (for peroxidases); i.e. good activity was observed only for substrates with more than one hydrogen bond donor/acceptor.},
  author       = {Ryabova, Ekaterina},
  isbn         = {91-7422-065-9},
  keyword      = {metabolism,Metabolism,enzymologi,Proteiner,enzymology,Proteins,hemoglobin,catalysis,peptide,peroxidase,protoporphyrin IX,Biokemi,Biochemistry},
  language     = {eng},
  pages        = {161},
  publisher    = {ARRAY(0x808dc40)},
  title        = {An Investigation of Peroxidase Activity in Biomimetic and Biological Systems},
  year         = {2004},
}