Vpliv kovinskih ionov na aktivnost katepsina K

diplomsko delo

Milica Janković (Avtor), Iztok Turel (Mentor)

Povzetek

Katepsin K, papainu podobna človeška peptidaza, je ključen encim pri razgradnji kolagena tipa I - glavne organske komponente, ki tvori kosti. Ker ima porušena kostna homeostaza za posledico nastanek številnih bolezni kot so osteoporoza, nekatere oblike artritisa in raka, je inhibicija encimske aktivnosti zaželena, kadar je aktivnost encima povezana z nastankom bolezni. Glede na to, da ima previsoka aktivnost katepsina K za posledico preveliko razgradnjo kosti, se razvijajo inhibitorji katepsina K za zdravljenje omenjenih bolezni. Trenutno je inhibicija katepsina K usmerjena na vezavo inhibitorjev v aktivno mesto encima, kar povzroči popolno izgubo encimske aktivnosti. Zaradi izgube drugih fizioloških funkcij aktivnega encima, ima popolna inhibicija aktivnosti katepsina K lahko tudi neželene stranske učinke. Zato bi bilo pomembno, da najdemo dovolj dobre delne inhibitorje encimske aktivnosti, ki bi v veliki meri inhibirali kolagenolitično aktivnost encima, ki je povezana s številnimi bolezni. V predstavljeni diplomski nalogi smo, v treh neodvisnih testih, določali vpliv in mehanizem delovanja izbranih kovinskih ionov (Ce4+, Ga3+, La3+ in Zn2+) na aktivnost katepsina K. Najprej smo vpliv in mehanizem delovanja izbranih kovinskih ionov na aktivnost encima določali s spremljanjem hidrolize sintetičnega substrata Z-FR-AMC. Ker smo želeli vse predvidene mehanizme inhibicije bolj natančno opredeliti, smo za vse izbrane kovinski ione pripravili diagrame specifičnih hitrosti, na osnovi katerih smo določili vrednosti parametrov Ki, α in β za vsak inhibitor. Dokazali smo da Ce4+, Ga3+ in La3+ delujejo kot hiperbolični oziroma delni inhibitorji, in sicer Ce4+ in La3+ po mehanizmu hiperbolične mešane inhibicije, Ga3+ pa kot hiperboličen kompetitivni inhibitor. Zn2+ ion se je izkazal kot predstavnik linearnih oziroma popolnih inhibitorjev encimske aktivnost, ki delujejo po mehanizmu linearne kompetitivne inhibicije. Ker rezultati, ki jih dobimo s kinetičnimi meritvami razgradnje sintetičnega substrata, ne veljajo nujno za naravne encimske substrate, smo naredili kvalitativno analizo kolagenolitične aktivnosti katepsina K v prisotnosti visokih koncentracij izbranih kovinskih ionov. Preverili smo tudi učinke inhibitorjev na encimsko aktivnost pri razgradnji azokazeina. Iz rezultatov slednjih poskusov smo ugotovili, da ne delujejo vsi inhibitorji enako dobro v prisotnosti različnih substratov. Na koncu diplomske naloge smo in situ pripravili komplekse derivatov bigvanida s Zn2+ ioni, ker smo želeli preveriti vpliv bigvanidov na inhibitorne učinke Zn2+ ionov. Rezultati poskusov kažejo na to, da se inhibitorna moč Zn2+ ionov na aktivnost katepsina K malenkost poveča, v kompleksu s fenforminom.

Ključne besede

encimi;katepsin K;inhibicija encimske aktivnosti;mehanizmi inhibicije;kinetične meritve;kovinski ioni;diplomska dela;

Podatki

Jezik:	Slovenski jezik
Leto izida:	2019
Tipologija:	2.11 - Diplomsko delo
Organizacija:	UL FKKT - Fakulteta za kemijo in kemijsko tehnologijo
Založnik:	[M. Janković]
UDK:	577.15:546.3/.9(043.2)
COBISS:	1538308035
Št. ogledov:	651
Št. prenosov:	162
Ocena:	0 (0 glasov)
Metapodatki:

Ostali podatki

Sekundarni jezik:	Angleški jezik
Sekundarni naslov:	Effects of metal ions on activity of cathepsin K
Sekundarni povzetek:	Cathepsin K, a papain-like human peptidase, is a key enzyme in the process of breakdown of type I collagen – the main bone-forming organic component. Since the disrupted bone homeostasis results in the emergence of numerous diseases such as osteoporosis, certain forms of arthritis and cancer, the inhibition of the enzyme activity is desirable when the enzyme activity is associated with the onset of the disease. Considering that the excessive activity of cathepsin K results in excessive bone breakdown, cathepsin K inhibitors are developed to treat the mentioned diseases. Currently, cathepsin K inhibition is directed towards the binding of inhibitors to the enzyme active site, leading to the complete loss of enzyme activity. Due to the loss of other physiological functions of the active enzyme, the complete inhibition of the activity of cathepsin K may also have undesirable side effects. Thus, it is important to find sufficiently good partial inhibitors of enzyme activity that would inhibit the collagenolytic activity of the enzyme associated with many diseases to a great extent. In the presented thesis, we determined the influence and mechanism of action of selected metal ions (Ce4+, Ga3+,La3+ and Zn2+) on the activity of cathepsin K in three independent tests. First, we determined the influence and mechanism of action of selected metal ions on enzyme activity by monitoring hydrolysis of synthetic substrate Z-FR-AMC. In order to confirm and more precisely define all of the selected inhibition mechanisms, specific velocity diagrams were prepared for all of the selected metal ions, based on which the values of kinetic parameters parameters were determined for each inhibitor. We showed that Ce4+, Ga3+ and La3+ act as hyperbolic or partial inhibitors, more specifically Ce4+ and La3+ by the mechanism of hyperbolic mixed inhibition, and Ga3+ as a hyperbolic competitive inhibitor. Zn2+ ion proved to be linear or complete inhibitors of enzymatic activity, acting by the mechanism of linear competitive inhibition. Since the results obtained with kinetic measurements of degradation of the synthetic substrate do not necessarily apply to natural enzyme substrates, we made a qualitative analysis of the collagenolytic activity of cathepsin K in the presence of high concentrations of selected metal ions. The effects of the inhibitors on the enzymatic activity during the degradation of azocasein were also examined. From the results of the latter experiments, it can be concluded that not all inhibitors act equally well in the presence of different substrates. At the end of this thesis we prepared in situ complexes of biguanide derivatives with Zn2+ ions, because we wanted to check the influence of biguanides on the inhibitory effects of Zn2+ ions. The results of the experiments indicated that the inhibitory effects of Zn2+ ions activity of cathepsin K slightly increased, in complex with phenformin.
Sekundarne ključne besede:	cathepsin K;enzyme inhibition;kinetic measurements;metal ions;
Vrsta dela (COBISS):	Diplomsko delo/naloga
Študijski program:	1000371
Konec prepovedi (OpenAIRE):	1970-01-01
Komentar na gradivo:	Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo
Strani:	29 str.
ID:	11214254