Enzimska karakterizacija piruvat-kinaze bakterije Bacillus subtilis

master's thesis

• Title (english): Enzymatic characterization of Bacillus subtilis pyruvate kinase
• Author: Kristina Kovač
• Mentor: Damjan Franjević (mentor)
• Mentor: Ivan Mijaković (sumentor)
• Committee member: Damjan Franjević (član povjerenstva)
• Committee member: Ivan Mijaković (član povjerenstva)
• Committee member: Zdravko Dolenec (član povjerenstva)
• Committee member: Branka Pevalek-Kozlina (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2012, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Piruvat-kinaza je ključni enzim glikolize i metabolizma ugljika. Katalizira završni korak glikolitičkog puta, prenoseći fosforilnu skupinu fosfoenolpiruvata (PEP) na ADP, stvarajući ATP i piruvat. Reakcija je ireverzibilna, te predstavlja važno regulatorno mjesto u glikolizi. U ovom istraživanju piruvat-kinaza je izolirana iz aerobne Gram-pozitivne bakterije Bacillus subtillis, te pročišćena metodom afinitetne kromatografije do homogenosti. Enzimska rekacija se odvijala pri pH 7.5, uz prisustvo supstrata (PEP i ADP), te iona - K+ i Mg2+ koji s ADP-om tvori Mg2+-ADP kompleks, koji djeluje kao pravi substrat enzimu. Cilj istraživanja je bio utvrditi kinetičke parametre – Km i Vm piruvat-kinaze. Eksperimentalni podaci dobiveni spektrofotometrijskom metodom prikazani su Michealis-Menten modelom enzimske kinetike, te su pomoću linearizacijskih prikaza izračunate vrijednosti Km=1,2138 mM, Vm=227,062 μmol NADH/min/μg proteina za ADP, te Km=0,9081 mM, Vm=97,1125 μmol NADH/min/μg proteina za PEP. Navedeni su rezultati dobiveni Hanes-Woolf linearizacijskim prikazom.

Abstract (english)

Pyruvate kinase is key enzyme in glycolysis and carbon metabolim. It catalyzes the final step of glycolytic pathway, transferring phosphoryl group of phosphoenolpyruvate (PEP) to ADP, yielding ATP and pyruvate. Reaction is irreversible and it represents important regulatory point in glycolysis. In this research pyruvate kinase is isolated from aerobic Gram-positive bacteria Bacillus subtilis and purified to homogenity. Enzymatic reaction proceeded at pH 7.5 in presence of substrates (ADP and PEP) and ions - K+ and Mg2+. Mg2+ creates a Mg2+-ADP complex with ADP, which acts as a true substrate of the enzyme. Goal of this research was to identify kinetic parameters - Km i Vm of pyruvate kinase. Experimental data given by spectrofotometric method are shown with Michaelis-Menten model of enzyme kinetics. By means of linear plotting, values were calculated to Km=1,2138 mM, Vm=227,062 μmol NADH/min/μg protein for ADP and Km=0,9081 mM, Vm=97,1125 μmol NADH/min/μg protein for PEP. Mentioned results were given by Hanes-Woolf linear plot.

• Keywords: piruvat-kinaza; enzimska karakterizacija; Bacillus subtilis
• Keywords (english): pyruvate kinase; enzymatic characterization; Bacillus subtilis
• Language: croatian
• URN:NBN: urn:nbn:hr:217:787265
• Study programme: Title: Biology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra edukacije biologije (magistar/magistra edukacije biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2018-04-18 11:34:27