Mehanizmi ostvarivanja supstratne specifičnosti u sintetskom i korektivnom mjestu izoleucil-tRNA-sintetaze

doctoral thesis

• Title (english): On the substrate specificity mechanisms of the synthetic and editing sites of isoleucyl-tRNA synthetase
• Author: Igor Živković
• Mentor: Ita Gruić-Sovulj (mentor)
• Committee member: Branimir Bertoša (predsjednik povjerenstva)
• Committee member: Mario Gabričević (član povjerenstva)
• Committee member: Zrinka Kovarik (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Chemistry); Zagreb
• Defense date and country: 2021-10-25, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Chemistry
• Universal decimal classification (UDC): 54 - Chemistry. Crystallography. Mineralogy

Abstract

Izoleucil-tRNA-sintetaza (IleRS) u sintetskom mjestu katalizira nastajanje Ile-tRNAIle za
biosintezu proteina. IleRS može pogrešno aminoacilirati i strukturno, odnosno kemijski, slične
nepripadne aminokiseline poput valina i norvalina što je toksično za stanicu. Kako bi popravila
vlastitu pogrešku, IleRS hidrolizira misaminoaciliranu tRNAIle u zasebnom, korektivnom
mjestu. U sklopu ove disertacije različitim kinetičkim metodama, uz široki spektar nepripadnih
aminokiselina, istražena je enzimska specifičnost sintetskog i korektivnog mjesta IleRS.
Sintetsko mjesto je uspješno diskriminiralo većinu testiranih aminokiselina u skladu s
njegovom visokom specifičnošću. No, iako je frekvencija nastajanja misaminoaciliranih tRNA
uz testirane aminokiseline bila izuzetno mala, sve misaminoacilirane tRNAIle bile su dobri
supstrati korektivnog mjesta. Dakle, suprotno od sintetskog, korektivno mjesto pokazuje široku
supstratnu specifičnost. Istovremeno, korektivno mjesto pokazuje visoku specifičnost u
sprečavanju hidrolize pripadne Ile-tRNAIle, što sugerira da je mehanizam negativne selekcije
usmjeravao evoluciju tog mjesta. Dodatna istraživanja su pokazala jedinstvene osobine
popravka pogreške kod IleRS u odnosu na njegove najbliže evolucijske srodnike.

Abstract (english)

Isoleucyl-tRNA synthetase (IleRS) whitin its synthetic site pairs isoleucine and tRNAIle for the
ribosomal protein syntesis. IleRS can also misaminoacylate tRNAIle with structurally and
chemically similar non-cognate valine and norvaline, which is toxic for the cell. To clear the
errors, IleRS hydrolyzes misaminoacylated tRNAIles at the dedicated editing site. Here we used
a broad spectrum of non-cognate amino acids to explore the mechanisms by which IleRS’
synthetic and editing sites exercise substrate specificity. We found that most of herein tested
amino acids were efficiently discriminated at the synthetic site in agrement with its known high
specificity. Nevertheless, despite the low misaminocylation frequencies, all misaminocylated
tRNAIles were rapidly hydrolyzed at the editing site. Thus, opposite to the synthetic site, the
editing site exhibits broad substrate specificity. At the same time, the editing site displays high
specificity against the cognate Ile-tRNAIle hydrolysis, indicating that negative selection directed
evolution of the editing site.

• Keywords: aminokiseline; enzimska specifičnost; korektivno mjesto; in trans popravak; izoleucil-tRNA-sintetaza; negativna selekcija; sintetsko mjesto
• Keywords (english): amino acids; corrective site; enzyme specificty; in trans editing; isoleucly-tRNA synthetase; negative selection; synthetic site
• Language: croatian
• URN:NBN: urn:nbn:hr:217:335823
• Promotion: 2021
• Study programme: Title: Doctoral study in chemistry; Study programme type: university; Study level: postgraduate; Academic / professional title: doktor/doktorica znanosti, područje prirodnih znanosti, polje kemija (doktor/doktorica znanosti, područje prirodnih znanosti, polje kemija)
• Type of resource: Text
• Extent: 137 str. ; 30 cm
• File origin: Born digital
• Access conditions: Open access
• Created on: 2021-11-04 14:42:29