Naslov
Serine-induced α-helix formation is essential for its productive positioning within atypical seryl- tRNA synthetase active site

Autori
Dulić, Morana ; Požar, Josip ; Weygand-Đurašević, Ivana ; Gruić Sovulj, Ita

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
23rd tRNA Workshop: From origin of life to biomedicine / Weil, Tobias ; Santos, Manuel A. S. - Aveiro, 2010

Skup
23rd tRNA Workshop : From origin of life to biomedicine

Mjesto i datum
Aveiro, Portugal, 28.01.2010. - 02.02.2010

Vrsta sudjelovanja
Poster

Vrsta recenzije
Nije recenziran

Ključne riječi
seryl-tRNA synthetase ; substrate ordering loop ; induced fit ; active site mutants

Sažetak
Seryl-tRNA synthetases (SerRS) are class II enzymes divided into two types: bacterial or canonical and methanogenic or atypical type. Recently determined crystal structure of atypical Methanosarcina barkeri SerRS (mMbSerRS) revealed that binding of serine is accompanied by a notable conformational change in the enzyme active site(1). This brings the prevously disordered serine ordering loop in an ordered conformation that consists of an N-terminal -helix and a loop in the C-terminal part. Observed induced fit rearrangements seem to be required for proper positioning of the carboxylate oxygen of serine (via direct contact with Gln400) for nucleophilic attack on the –phosphate of ATP. Binding of serine onto enzyme was followed by isothermal titration calorimetry (ITC). Parameters determined for wild-type enzyme clearly show that this interaction is enthalpically driven. The interactions involved in protein-ligand association as well as ones in α-helix formation outcome the entropically unfavorable reduction of randomness of the system. We have designed several mutants bearing mutations in serine ordering loop in order to determine its role in serylation of tRNASer by mMbSerRS. The rationale was to either change the secondary structure of the loop or to affect specific contacts with the serine. Mutants were kinetically and thermodynamically characterized. Formation of an α-helix and its orientation is crucial for productive positioning of serine as concluded from order of magnitude reduced kcat values. On the other side, mutation designed to provoke formation of an -helix in the C-terminal part of the serine ordering loop, significantly decreases enzyme's afinity toward serine. Surprisingly, mutation of Gln400 which is in direct contact with the substrate has only slight effect on enzyme's efficiency both in activation and in aminoacylation, as well as on binding. On the other hand, substitution of His250 that is only in indirect contact with serine mediated precisely by Gln400, abolishes enzymatic activity in aminoacylation, but has almost no effect on binding and activation of serine. These results, together with gel-mobility shift analysis suggest that His250 is important in one of the steps involving tRNA.

Izvorni jezik
Engleski

Znanstvena područja
Kemija, Biologija

POVEZANOST RADA