The Mechanism of Peptide Hydrolysis Catalysed by Dipeptidyl Peptidase III from Bacteroides thetaiotaomicron

Dipeptidyl peptidase III (DPP III) is a zinc- dependent peptidase that cleaves dipeptides off of N-termini of its substrates. Previous studies on human DPP III reveal a reaction mechanism similar to that of thermolysin. Since the active site is conserved within the DPP III family, it is not surprising that the mechanism determined for Bacteroides thetaiotaomicron DPP III (BtDPP III) closely resembles that of hDPP III. However, the hydrogen bond network within the model differs slightly from that in the human ortholog, which results in two proposed pathways. The calculated Gibbs activation energy of 90.1 kJ mol–1 is larger than the one calculated from kinetic data for the preferred substrate Arg2-2-naphthylamide at room temperature (69 kJ mol–1), suggesting the importance of treating the whole DPP III enzyme in the calculations

Demystifying DPP III Catalyzed Peptide Hydrolysis-Computational Study of the Complete Catalytic Cycle of Human DPP III Catalyzed Tynorphin Hydrolysis

Dipeptidyl peptides III (DPP III) is a dual-domain zinc exopeptidase that hydrolyzes peptides of varying sequence and size. Despite attempts to elucidate and narrow down the broad substrate- specificity of DPP III, there is no explanation as to why some of them, such as tynorphin (VVYPW), the truncated form of the endogenous heptapeptide spinorphin, are the slow-reacting substrates of DPP III compared to others, such as Leu- enkephalin. Using quantum molecular mechanics calculations followed by various molecular dynamics techniques, we describe for the first time the entire catalytic cycle of human DPP III, providing theoretical insight into the inhibitory mechanism of tynorphin. The chemical step of peptide bond hydrolysis and the substrate binding to the active site of the enzyme and release of the product were described for DPP III in complex with tynorphin and Leu-enkephalin and their products. We found that tynorphin is cleaved by the same reaction mechanism determined for Leu- enkephalin. More importantly, we showed that the product stabilization and regeneration of the enzyme, but not the nucleophilic attack of the catalytic water molecule and inversion at the nitrogen atom of the cleavable peptide bond, correspond to the rate-determining steps of the overall catalytic cycle of the enzyme

Modification of fly ash for photocatalytic polymer nanocomposites immobilization

Leteći pepeo (FA) modificiran je različitim postupcima: klorovodičnom kiselinom te duljim termičkim tretiranjem kao i sumpornom kiselinom uz prisutnost tetraetilortosilikata (TEOS) i polietilenglikola (PEG).Tako pripremljenim uzorcima modificiranog letećeg pepela dodane su nanočestice čistog titanijeva dioksida (TiO2 ) u iznosu od 10 mas% čime je pripremljena druga serija fotokatalizatora (FA-Ti). Različitim modifikacijama dolazi do promjene specifične površine i volumena pora čestica letećeg pepela, stoga su uzorci karakterizirani BET metodom. Zatim im je karakterizran izgled i struktura čestica letećeg pepela pretražnim elektronskim mikroskopijom (SEM). Za sve uzorke provedena je ocjena njihove fotokatalitičke aktivnosti pa je u tu svrhu pripremljena vodena otopina onečišćena bojilom RR45. Efikasnost fotokatalitičke razgradnje provedena je nakon postupaka adsorpcije bojila na fotokatalizatore tijekom stajanja u mraku 60 minuta. Fotokatalitička aktivnost određena je tako da je modelna otopina izložena UV zračenju i svakih 15 minuta, u vremenskom periodu od 60 minuta, uzimani su alikvoti uzoraka onečišćene vode i određivana im je apsorbancija na UV/VIS spektrofotometru. Iz rezultata vidljivo je da uzorci čistog modificiranog letećeg pepela nisu povećali fotokatalitičku aktivnost pepela, za razliku od kompozitnih uzoraka, modificiranog letećeg pepela i titanijeva dioksida, gdje je došlo do znatnog unaprijeđenja razgradnje bojila RR45.Fly ash (FA) has been modified by various methods: by hydrochloric acid with longer thermal treatment and by sulfuric acid in the presence of tetraethylorthosilicate (TEOS) and polyethylene glycol (PEG). In thus prepared samples of modified fly ash nanoparticles of pure titanium dioxide (TiO) in the amount of 10% by weight were added and new series of photocatalysts (FA-Ti) were prepared. Various modifications iniciated change of specific surface area and pore volume of the fly ash particles because of those samples were characterized by BET method. Then, morfology and structure of the fly ash particles were characteried by scanning electron microscopy (SEM). For all samples, validation of their photocatalytic activity had been done, and for this purpose aqueous solution contaminated by dye RR45 was prepared. The efficiency of photocatalytic activites was carried out after the adsorption procedures of dyes on photocatalysts during 60 minutes staing in the dark. The photocatalytic activity is determined in the way that the model solutions was exposed to UV light and every 15 minutes, for a time period of 60 minutes, aliquots samples of contaminated water were taken and their absorbance is determined by UV / VIS spectrophotometer. From the results it is evident that modified fly ash samples does not improved photocatalytic activity of the ash, in contrast to the composite samples, the modified fly ash and titanium dioxide, which show significant improvement of the photocatalytic decomposition of dyes RR45

Modification of fly ash for photocatalytic polymer nanocomposites immobilization

Leteći pepeo (FA) modificiran je različitim postupcima: klorovodičnom kiselinom te duljim termičkim tretiranjem kao i sumpornom kiselinom uz prisutnost tetraetilortosilikata (TEOS) i polietilenglikola (PEG).Tako pripremljenim uzorcima modificiranog letećeg pepela dodane su nanočestice čistog titanijeva dioksida (TiO2 ) u iznosu od 10 mas% čime je pripremljena druga serija fotokatalizatora (FA-Ti). Različitim modifikacijama dolazi do promjene specifične površine i volumena pora čestica letećeg pepela, stoga su uzorci karakterizirani BET metodom. Zatim im je karakterizran izgled i struktura čestica letećeg pepela pretražnim elektronskim mikroskopijom (SEM). Za sve uzorke provedena je ocjena njihove fotokatalitičke aktivnosti pa je u tu svrhu pripremljena vodena otopina onečišćena bojilom RR45. Efikasnost fotokatalitičke razgradnje provedena je nakon postupaka adsorpcije bojila na fotokatalizatore tijekom stajanja u mraku 60 minuta. Fotokatalitička aktivnost određena je tako da je modelna otopina izložena UV zračenju i svakih 15 minuta, u vremenskom periodu od 60 minuta, uzimani su alikvoti uzoraka onečišćene vode i određivana im je apsorbancija na UV/VIS spektrofotometru. Iz rezultata vidljivo je da uzorci čistog modificiranog letećeg pepela nisu povećali fotokatalitičku aktivnost pepela, za razliku od kompozitnih uzoraka, modificiranog letećeg pepela i titanijeva dioksida, gdje je došlo do znatnog unaprijeđenja razgradnje bojila RR45.Fly ash (FA) has been modified by various methods: by hydrochloric acid with longer thermal treatment and by sulfuric acid in the presence of tetraethylorthosilicate (TEOS) and polyethylene glycol (PEG). In thus prepared samples of modified fly ash nanoparticles of pure titanium dioxide (TiO) in the amount of 10% by weight were added and new series of photocatalysts (FA-Ti) were prepared. Various modifications iniciated change of specific surface area and pore volume of the fly ash particles because of those samples were characterized by BET method. Then, morfology and structure of the fly ash particles were characteried by scanning electron microscopy (SEM). For all samples, validation of their photocatalytic activity had been done, and for this purpose aqueous solution contaminated by dye RR45 was prepared. The efficiency of photocatalytic activites was carried out after the adsorption procedures of dyes on photocatalysts during 60 minutes staing in the dark. The photocatalytic activity is determined in the way that the model solutions was exposed to UV light and every 15 minutes, for a time period of 60 minutes, aliquots samples of contaminated water were taken and their absorbance is determined by UV / VIS spectrophotometer. From the results it is evident that modified fly ash samples does not improved photocatalytic activity of the ash, in contrast to the composite samples, the modified fly ash and titanium dioxide, which show significant improvement of the photocatalytic decomposition of dyes RR45

Multi scale computational approaches to understand a structure, dynamics and activity of thehuman dipeptidyl-peptidase III

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

The first dipeptidyl peptidase III from a thermophile: Structural basis for thermal stability and reduced activity

Dipeptidyl peptidase III (DPP III) isolated from the thermophilic bacteria Caldithrix abyssi (Ca) is a two-domain zinc exopeptidase, a member of the M49 family. Like other DPPs III, it cleaves dipeptides from the N-terminus of its substrates but differently from human, yeast and Bacteroides thetaiotaomicron (mesophile) orthologs, it has the pentapeptide zinc binding motif (HEISH) in the active site instead of the hexapeptide (HEXXGH). The aim of our study was to investigate structure, dynamics and activity of CaDPP III, as well as to find possible differences with already characterized DPPs III from mesophiles, especially B. thetaiotaomicron. The enzyme structure was determined by X-ray diffraction, while stability and flexibility were investigated using MD simulations. Using molecular modeling approach we determined the way of ligands binding into the enzyme active site and identified the possible reasons for the decreased substrate specificity compared to other DPPs III. The obtained results gave us possible explanation for higher stability, as well as higher temperature optimum of CaDPP III. The structural features explaining its altered substrate specificity are also given. The possible structural and catalytic significance of the HEISH motive, unique to CaDPP III, was studied computationally, comparing the results of long MD simulations of the wild type enzyme with those obtained for the HEISGH mutant. This study presents the first structural and biochemical characterization of DPP III from a thermophile

Chemical composition of aronia berry wine

U novije vrijeme zabilježen je rastući interes za konzumacijom funkcionalne hrane koja svojim sastavom pozitivno utječe na razvoj i funkcioniranje ljudskog organizma. Plodovi aronije (Aronia melanocarpa) i proizvodi dobiveni njihovom preradom predstavljaju vrijedan izvor polifenola, posebno proantocijanidina i antocijana. Zbog visoke zastupljenosti u plodovima voća i visoke antioksidativne aktivnosti, polifenoli se smatraju najznačajnijim dijetarnim antioksidansima. Antioksidansi su tvari koje usporavaju procese oksidacije u organizmu, te na taj način usporavaju procese starenja. Cilj ovog istraživanja je bio utvrditi kemijski sastav, koncentraciju ukupnih i pojedinačnih antocijana te antioksidacijski kapacitet voćnih vina od aronije proizvedene u kontinentalnoj Hrvatskoj.Recently there is a growing interest in consumption of functional food whose composition has a positive effect on the development and functioning of human organism. Chokeberry (Aronia melanocarpa) and derived products represent a valuable source of polyphenols, specifically proanthocyanidins and anthocyanins. Due to high representation in fruits and high antioxidant activity, polyphenols are considered to be the most important dietary antioxidants. Antioxidants are substances that delay the oxidation processes in the body, thereby slowing down the aging process. The aim of this study was to determine the chemical composition, total anthocyanin content, amount of single anthocyanins and antioxidant capacity of chokeberry fruit wines produced in continental part of Croatia