Regulatory role of L-type pyruvate kinase N-terminal domain
Date
2013-11-05
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Abstract
Püruvaat on oluline metabolismi substraat ja seetõttu on püruvaadi kinaaside aktiivsuse regulatsioon oluline raku energia- ja süsinikuvoogude suunamise seisukohast. Maksas, kus samaaegselt toimivad need mõlemad metabolismi rajad, esineb koe-spetsiifiline püruvaadi kinaaas (L-PK), mis on aktiveeritud homotroopselt selle ensüümi substraadi fosfoenoolpüruvaadi (PEP) poolt ning heterotroopselt fruktoos-1,6-bisfosfaadi (FBP) poolt. Samas on need mõlemad efektid omakorda reguleeritud selle ensüümi N-terminaalse domeeni fosforüleerimise kaudu. Käesolevas töös uuriti selle regulatoorse fosforüleerimise nähtuse tagamaid, kasutades selleks klassikalisi valgukeemia meetodeid ning keemilise kineetika rakendusi koos ligandide seostumise modelleerimisega arvutil.
Töö tulemused näitasid, et mitte-fosforüülitud L-PK poolt katalüüsitud reaktsioon on PEP korral iseloomustatud tavalise hüperboolse Michelis-Menteni reaktsioonikiiruse võrrandiga (KPEP = 0.11 mM). Samas lülitas cAMP-sõltuva proteiinkinaasi poolt katalüüsitud valgu fosforüülimine sisse L-PK kooperatiivsuse PEP suhtes ning ka selle ensüümi aktiivsuse allosteerilise regulatsiooni. Nii iseloomustas stöhhiomeetriliselt fosforüleeritud ensüümi, kus ühe valgu tetrameerse molekuli kohta esines neli fosfaatrühma, KPEP väärtus 2.2 mM ja Hilli koefitsient n = 2.5. Samuti ilmnes, et kooperatiivsed omadused lülitusid sisse tetrameerse valgu esimese subühiku fosforüleerimisel, kusjuures edasine fosfaatrühmade lisamine ainult moduleeris seda efekti. Samuti leiti, et mitte-fosforüülitud L-PK aktiivsust ei mõjutanud ka allosteeriline aktivaator FBP. Kuna fosforüleeritud L-PK aktiivsus suureneb oluliselt allosteerilise aktivaatori FBP juuresolekul, siis järeldati, et fosforüleerimine toimib ka selle ensüümi allosteerilise regulatsiooni molekulaarse lülitina.
Regulatoorne fosforüülimine toimub seriini nr 12 juures, mis asub N-terminaalses valgujärjestuses MEGPAGYLRR10AS12VAQLTQEL20GTAFF. Selle valgujärjestuse rolli selgitamiseks uuriti fosforüülitava seriini ümbruses asuvate aminohapete punktmutatsioonide mõju valgu katalüütilistele omadustele. Muutused olid positsioonides 9, 10 ja 13 ning asenduseks kasutati aminohappeid A, L, Q ja E, mis võimaldas muuta selle domeeni summaarset laengut ja uurida, kas ioonsete rühmade ümberjaotumine ja uute ioonsete rühmade sisseviimine võib imiteerida seriini jäägi fosforüülimise efekti. Ilmnes, et mõned asendused mõjutasid valgu aktiivsust, vähendades selle afiinsust PEP suhtes ning sellega imiteerisid fosforüülimist. Saadud tulemus on kooskülas A. Fentoni ja kaastöötajate poolt pakutud oletusega (Fenton & Tang, 2009; Holyoak et al., 2013), et N-domeen võib seostuda valgumolekuli mingis muus piirkonnas ja selliselt mõjutada substraadi sidumist. Selle sisemolekulaarse kompleksi lõhub aga domeeni fosforüülimine. Samas aga ilmnes tõsiasi, et N-domeeni struktuuri ja kogulaengu muutmine ei indutseeri ensüümi kooperatiivsust PEP suhtes.
Lähtudes oletusest, et N-domeen võib tänu oma paindlikkusele toimida valgumolekuli teiste osadega, teostati arvutil modelleerimise teel võimalike sidumiskohtade otsing. Selleks uuriti regulatoorse domeeni fragmendi RRASVA ja selle fosforüülitud analoogi RRAS(Pi)VA seostumist L-PK alaühikuga. Leiti, et RRASVA seostumine võib toimuda ensüümi aktiivtsentri piirkonnas ning see võib tõepoolest olla oluline PEP sidumise seisukohast. Samal ajal toimus fosfopeptiidi sidumine subühiku C-domeenil, kus seostub allosteeriline ligand FBP. Seega võib sisemolekulaarsete komplekside teke tõepoolest nihutada tasakaalu ensüümi aktiivse R-vormi ja vähemaktiivse T-vormi vahel. Samas võib kooperatiivsuse sisselülitamine toimuda fosforüleeritud N-domeeni seostumisel L-PK allosteerilises tsentris.
Molekulaarne lülitusmehhanism allosteerilise ja mitte-allosteerilise L-PK vormide vahel, mis põhineb regulatoorse domeeni fosforüülimisel, võib omada olulist tähtsust maksas toimuva glükoosi metabolism mõistmisel.
Pyruvate is an essential substrate in metabolic processes, and regulation of the activity of pyruvate kinases is essential for control over the energy and carbon fluxes in living cells. In liver tissue, where both of these glycolysis and glyconeogenesis pathways are in function, the tissue-specific pyruvate kinase (L-PK) is activated homotropically by phosphoenolpyruvate (PEP) and heterotropically by fructose-1,6-bisphosphate (FBP), and both of these effects depend on phosphorylation of the N-terminal domain of the enzyme. In this dissertation the regulatory role of the N-terminal domain has been studied by using conventional methods of protein chemistry and chemical kinetics along with computational ligand docking analysis. The results of this study revealed that the activity of the non-phosphorylated L-PK was characterised by a common hyperbolic Michaelis-Menten plot in the case of PEP (KPEP = 0.11 mM) and the activity of the enzyme was not regulated by FBP. The cooperativity of the enzyme for PEP was switched on by phosphorylation of the protein by cAMP-dependent protein kinase. The stoichiometrically phosphorylated enzyme, containing four moles of phosphate per tetrameric L-PK molecule, was characterised by KPEP = 2.2 mM and the Hill coefficient n = 2.5. It was also observed that enzyme cooperativity was mostly engaged by phosphorylation of the first subunit in the tetrameric protein, while further phosphorylation only modulated this effect. Further, it was found that the activity of the non-phosphorylated L-PK was not elevated by FBP, and this ligand acted as a relatively weak reversible inhibitor. As the phosphorylated L-PK is a subject of significant allosteric activation by FBP, it was concluded that phosphorylation should also function as a molecular switch in the case of the allosteric properties of L-PK. The regulatory phosphorylation occurs in the serine residue in position 12 of the N-terminal sequence MEGPAGYLRR10AS12VAQLTQEL20GTAFF, and in this study the influence of point mutations around the phosphorylation site in positions 9, 10 and 13 on the catalytic properties of the mutants was studied. Amino acids A, L, Q and E were introduced into these positions to alter the net charge of the regulatory domain and to check whether redistribution and/or introduction of new ionic groups can mimic the effect of serine phosphorylation. It was found that some of these mutations affected the catalytic activity of the enzyme by reducing the effectiveness of phosphoenolpyruvate binding, simulating thus the effect of regulatory phosphorylation. This result was in agreement with the ideas suggested by A. Fenton & Tang (Fenton & Tang, 2009) that the flexible N-domain may interact with the main body of the enzyme affecting substrate binding, and this interaction is interfered with by phosphorylation. However, differently from phosphorylation, the cooperativity of the enzyme was not switched on by these mutations in the N-domain. Finally, following the suggestion that the N-domain interacts with the main body of the L-PK molecule, computational analysis was made to identify putative binding sites. The docking site of the N-domain peptide RRASVA was found in the vicinity of the enzyme active centre, while docking of the phosphorylated analogue RRAS(Pi)VA occurred on the C domain of the L-PK molecule, in a site overlapping with the allosteric site for FBP binding. This means that formation of these intramolecular complexes may indeed change equilibrium between the active R-state and the less active T-state of the enzyme, while induction of cooperativity by the N-domain phosphorylation could be connected with phosphopeptide intramolecular binding with the enzyme allosteric site. The phenomenon of switching between non-allosteric and allosteric forms of L-PK and the possibility of modulating of the enzyme allostery by phosphorylation may have some implication for further understanding of the regulation of glycolysis in the liver.
Pyruvate is an essential substrate in metabolic processes, and regulation of the activity of pyruvate kinases is essential for control over the energy and carbon fluxes in living cells. In liver tissue, where both of these glycolysis and glyconeogenesis pathways are in function, the tissue-specific pyruvate kinase (L-PK) is activated homotropically by phosphoenolpyruvate (PEP) and heterotropically by fructose-1,6-bisphosphate (FBP), and both of these effects depend on phosphorylation of the N-terminal domain of the enzyme. In this dissertation the regulatory role of the N-terminal domain has been studied by using conventional methods of protein chemistry and chemical kinetics along with computational ligand docking analysis. The results of this study revealed that the activity of the non-phosphorylated L-PK was characterised by a common hyperbolic Michaelis-Menten plot in the case of PEP (KPEP = 0.11 mM) and the activity of the enzyme was not regulated by FBP. The cooperativity of the enzyme for PEP was switched on by phosphorylation of the protein by cAMP-dependent protein kinase. The stoichiometrically phosphorylated enzyme, containing four moles of phosphate per tetrameric L-PK molecule, was characterised by KPEP = 2.2 mM and the Hill coefficient n = 2.5. It was also observed that enzyme cooperativity was mostly engaged by phosphorylation of the first subunit in the tetrameric protein, while further phosphorylation only modulated this effect. Further, it was found that the activity of the non-phosphorylated L-PK was not elevated by FBP, and this ligand acted as a relatively weak reversible inhibitor. As the phosphorylated L-PK is a subject of significant allosteric activation by FBP, it was concluded that phosphorylation should also function as a molecular switch in the case of the allosteric properties of L-PK. The regulatory phosphorylation occurs in the serine residue in position 12 of the N-terminal sequence MEGPAGYLRR10AS12VAQLTQEL20GTAFF, and in this study the influence of point mutations around the phosphorylation site in positions 9, 10 and 13 on the catalytic properties of the mutants was studied. Amino acids A, L, Q and E were introduced into these positions to alter the net charge of the regulatory domain and to check whether redistribution and/or introduction of new ionic groups can mimic the effect of serine phosphorylation. It was found that some of these mutations affected the catalytic activity of the enzyme by reducing the effectiveness of phosphoenolpyruvate binding, simulating thus the effect of regulatory phosphorylation. This result was in agreement with the ideas suggested by A. Fenton & Tang (Fenton & Tang, 2009) that the flexible N-domain may interact with the main body of the enzyme affecting substrate binding, and this interaction is interfered with by phosphorylation. However, differently from phosphorylation, the cooperativity of the enzyme was not switched on by these mutations in the N-domain. Finally, following the suggestion that the N-domain interacts with the main body of the L-PK molecule, computational analysis was made to identify putative binding sites. The docking site of the N-domain peptide RRASVA was found in the vicinity of the enzyme active centre, while docking of the phosphorylated analogue RRAS(Pi)VA occurred on the C domain of the L-PK molecule, in a site overlapping with the allosteric site for FBP binding. This means that formation of these intramolecular complexes may indeed change equilibrium between the active R-state and the less active T-state of the enzyme, while induction of cooperativity by the N-domain phosphorylation could be connected with phosphopeptide intramolecular binding with the enzyme allosteric site. The phenomenon of switching between non-allosteric and allosteric forms of L-PK and the possibility of modulating of the enzyme allostery by phosphorylation may have some implication for further understanding of the regulation of glycolysis in the liver.
Description
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Keywords
püruvaat, kinaasid, fosforüülimine, regulatsioon, modelleerimine (teadus), pyruvate, kinases, phosphorylation, regulation, modelling (science)