Sažetak Dipeptidil-peptidaza III (DPP III) je o cinku ovisna egzopeptidaza koja katalizira reakciju hidrolize peptidne veze odcjepljujući dipeptid s N-kraja svojih supstrata. U okviru ove doktorske ...disertacije detaljno je istražena struktura i dinamika ljudske DPP III i njenih kompleksa primjenom računalnih pristupa različitih stupnjeva složenosti. Modeliranje je bilo temeljeno na trodimenzionalnim strukturama enzima određenim rentgenskom strukturnom analizom. Molekulsko-dinamičke (MD) simulacije slobodnog (bez vezanog liganda) enzima otkrile su veliku fleksibilnost proteina, a simulacije kompleksa DPP III sa sintetskim i peptidnim supstratima pridonijele su razumijevanju široke supstratne specifičnosti ovoga enzima, te u kombinaciji s računom slobodne energije omogućile određivanje najvjerojatnijeg i kemijski aktivnog načina vezanja supstrata. Aktivna konformacija enzima dodatno je potvrđena primjenom različitih MD metoda i hibridnim kvantno-mehaničkim molekulskomehaničkim (QM/MM) računima različitih načina koordinacije cinka. Mehanizam reakcije hidrolize određen je na modelnom sustavu sastavljenom od supstrata, aminokiselinskih ostataka koji neposredno sudjeluju u procesu katalize i ostataka koji koordiniraju ion cinka.
The dipeptidyl-peptidase III (DPP III) is a zinc-exopeptidase that hydrolyzes dipeptide from the N-terminus of its substrates. In this thesis a structure and dynamics of ligand-free enzyme and its complexes is investigated in detail by multi scale computational approaches. Modeling study was based on the three-dimensional enzymes structures obtained by X-ray diffraction analysis. Molecular dynamics (MD) simulations of the ligand-free enzyme revealed large flexibility of the protein, while the simulations of DPP III in the complexes with synthetic and peptide substrates enabled understanding of its broad substrate specificity, and, in combination with the free energy calculations, helped in determination of the most probable and chemically active ligand binding mode. The active enzyme conformation was confirmed by different MD approaches as well as by the hybrid quantum mechanicsmolecular mechanics (QM/MM) calculation of the different zinc ion coordinations. The reaction mechanism was determined using the model system consisting of substrate, amino acids participating in catalysis and the residues that coordinate the zinc ion.
Tiosemikarbazoni mogu postojati u nekoliko tautomernih oblika i konformacija, s mogućnošću stvaranja intra- i intermolekulskih vodikovih veza. Detaljna strukturna karakterizacija tiosemikarbazona, ...kao spojeva koji se povezuju sa širokim spektrom bioloških aktivnosti, preduvjet je za bolje razumijevanje njihove bioaktivnosti, kao i dobivanje molekula s poboljšanim farmakološkim svojstvima. Primjenom višenuklearne spektroskopije NMR (1H, 13C i 15N) u čvrstom stanju i otopini istražen je utjecaj supstituenata, prirode otapala (različitih polarnosti, odnosno različitih proton-donorskih i proton-akceptorskih svojstava) i temperature na molekulsku konformaciju, tautomeriju i prirodu vodikovih veza (intra- i intermolekulskih) derivata salicilaldehid i 2-metoksibenzaldehid tiosemikarbazona. Kombinacija poznate kristalne strukture te izmjerenih kemijskih pomaka 13C i 15N u čvrstom stanju, korištena je kao temelj za detekciju strukturnih promjena u otopini. Eksperimentalni rezultati upotpunjeni su računalnim istraživanjem konformacijskog prostora odabranih spojeva u vakuumu, i organskim otapalima (PCM pristup), kombinacijom molekulsko- i kvantno-mehaničkih metoda. Najstabilnije strukture, dobivene molekulskom mehanikom, reoptimirane su u vakuumu, CHCl3 i DMSO primjenom računskog modela B3LYP-D3/6- 311++G(2df,2p). Metodom populacijske analize QTAIM, korištenjem Koch-Popelierovih kriterija, okarakterizirane su intramolekulske vodikove veze u najstabilnijim konformerima istraživanih spojeva.
Thiosemicarbazones can exist in several tautomeric forms and conformations, with the possibility of intra- and intermolecular hydrogen bonding. Detailed structural characterization of thiosemicarbazones, as compounds that are associated with a wide spectrum of biological activities, is a prerequisite for better understanding of their bioactivity and for obtaining molecules with improved pharmacological properties. Solid and solution state multinuclear NMR spectroscopy (1H, 13C i 15N) was emloyed to explore the influence of the substituents, the nature of the solvent (different polarity and different proton-donor and proton-acceptor abbilities) and temperature on the molecular conformation, tautomerism and the nature of hydrogen bonding (intra- and intermolecular) of salicylaldehyde and 2-methoxybenzaldehyde thiosemicarbazone derivatives. The combination of known crystal structures and observed 13C and 15N chemical shifts in the solid state, was used as a basis for the detection of structural changes in solution. The experimental results were complemented with computational research of conformational space of selected compounds in a vacuum, and organic solvents (PCM approach), by combination of molecular- and quantum-mechanical methods. The most stable structures, obtained by molecular mechanics, were reoptimized in vacuum, CHCl3 and DMSO with B3LYP-D3/6-311++G(2df,2p) computational model. QTAIM population analysis method, together with Koch-Popelier criteria, was used for characterization of intramolecular hydrogen bonds in most stable conformers of investigated compounds.
