Modification of the activity of hydrolytic enzymes by the nature of the non-endogenous/non-native metal ions

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Magyar nyelvű absztrakt

Összefoglaló Zeyad Hasan Abdullah Nafaee "Hidrolitikus enzimek aktivitásának módosítása nem endogén/idegen fémionok révén" c. PhD disszertációjához. A PhD munka kétféle hidrolitikus enzim vizsgálatát tűzte ki célul, különös tekintettel a fémionok katalitikus aktivitásra gyakorolt hatására. A TEM-1 β-laktamáz, a β-laktám antibiotikumok hidrolízisét segíti elő, azokkal szemben rezisztenciát okozva. Az NColE7 a vetélytárs baktériumok DNS-ének hidrolízise révén védi meg a gazdasejtet. A TEM-1 β-laktamáz nem metalloenzim, de számos potenciális fémkötőhely található benne. A fehérjetisztítás sikere fémion-affinitás kromatográfiával bizonyította a Ni(II) ionok kölcsönhatását a felszíni hisztidin oldalláncokkal. Ezt spektroszkópiás vizsgálatok is alátámasztották. A fehérjében található kén donoratomok szoft jellegű fémionokat köthetnek meg, mint a Cd(II), vagy Hg(II) ionok. Míg a Hg(II) ionok csökkentették az enzim katalitikus aktivitását, a Ni(II) és Cd(II) ionok elősegítették a szubsztrát hidrolízisét. A fémion természete meghatározó: a Hg(II) ionok az aktív központ közelében kötődve gátolták a reakció végbemenetelét, míg a másik két ion a szubsztrátot aktiválta. Az enzimkinetika felülvizsgálatára is sor került. Az NColE7 metalloenzim aktív központjában egy cinkion kötődik három hisztidin oldallánchoz és a szubsztrát hasítandó csoportjához, tetratéderes elrendeződésben. A rögzített geometria miatt a vizsgált Ni(II), Cd(II) és Cu(II), idegen fémionok kötődését a fehérjéhez csak annak apo (fémmentes) formájában mutattuk ki. Kompetitív körölmények között a fehérje cink-kötött formában van. Az eddigi ismeretektől eltérően, úgy találtuk, hogy a fehérje fémion távollétében is működőképes. Ugyanakkor, a Ni(II) ion lényegesen megnöveli a katalitikus aktivitást, valószínűleg a folyamat mechanizmusának megváltoztatásával. Ez a fémion a fehérje és a szubszrát mellett egy vízmolekulát is megköthet, amelyet aktiválva hatékony nukleofil támadó ágens jön létre. A tanulmányozott enzimek a bakteriális védekezőrendszer részét képezik, így reményeink szerint, eredményeink hozzájárulnak hatékonyabb antibiotikumok kifejlesztéséhez.

Absztrakt (kivonat) idegen nyelven

The research work included in this dissertation involved the investigation of two types hydrolytic enzymes. TEM-1 β-lactamase was studied with the emphasis of its interaction with non-endogeneous, toxic metal ions (Ni(II), Cd(II) and Hg(II)) and their effect on the catalytic activity of the enzyme. This bacterial enzyme can hydrolyze the β-lactam ring, providing resistance against β-lactam antibiotics. As such it is a frequent target of the researches related to the development of antibiotics resistance posing a serious health threat in humans. TEM-1 β-lactamase is not a metalloenzyme, but it possesses several putative metal ion binding sites, suggesting that it may interact with metal ions affecting its function. The most important achievements of this topic were the following. We have successfully expressed TEM-1 β-lactamase from the modified pET-21a(+) vector carrying the coding sequence of the DNA binding domain of the His-tagged Nuclear Factor I, in spite of the fact that the gene of TEM-1 β-lactamase is located outside of the multi-cloning site. We purified the protein from the bacterial pellets in its native fold by immobilized metal ion chromatography (IMAC), based on the surface histidine pairs as potential metal binding sites. Our strategy involved two steps of IMAC and single step of anion exchange, providing a yield of 1.9 mg/g of wet bacterial pellet weight, which is comparable to previous purification methods carried out under denaturing conditions. We could identify the TEM-1 β-lactamase protein by mass spectrometric investigation of the trypsine digested fragments, as well as by intact protein molecular mass measurement. The results proved that purified TEM-1 β-lactamase was a mature enzyme (24-286 a.a.), in which the 23 a.a. signal sequence – required for the export of the protein to periplasm – was already cleaved. In addition, we could identify a mutation of the amino acid in position 82, which proved to be an isoleucine instead of the expected valine in the E.coli TEM-1 β-lactamase. The MS results also proved that the two Cys residues (Cys75 and Cys121) were oxidized in a form of a disulfide bridge. Analysing the CD spectrum of TEM-1 β-lactamase in solution we could show that he obtained secondary structure composition i.e., the percentage of α-helices, β-sheets, turns, and other conformations is close to that calculated from the published crystal structure. Thus, TEM-1 β-lactamase was folded in solution into its functional structure. We could demonstrate by CD spectroscopy, that the secondary structure of TEM-1 β-lactamase changed only slightly upon interaction with the selected Ni(II), Cd(II) and Hg(II) ions. Since the interaction with Ni(II) was already proven by the success of the IMAC purification, this supports the binding of Ni(II) and also Cd(II) ion to the surface histidine pairs that are at suitable distance for chelation. The most significant (~20%) decrease of the α-helix content was observed upon increase of the Hg(II):protein concentration ratio up to 10:1. This suggested that Hg(II) is most probably bound close to the methionine thioether groups inside the protein structure. We have identification the Ni(II) binding sites in TEM-1 β-lactamase. The slight but continuous change of the CD spectra upon increasing the metal ion content did not allow for determination of the number of the bound metal ions. It rather suggested that the enzyme could most likely bind more than one metal ion with not too high affinity. Therefore, mass spectrometric experiments were used to prove that all the studied Ni(II):enzyme molar ratio (2:1-10:1), the major peak was assigned to the monometallated TEM-1 β-lactamase. The relative intensity of the peaks corresponding to the two or three Ni(II)-chelating protein molecules, only slightly increased upon increasing the metal ion excess. This strongly suggested that the sidechain donor atoms of His151 and His156 being at ~3.5 Å distance, are ideal for metal ion binding. Furthermore, Asn152 and Asp155 amino acid residues in their close vicinity may strengthen this interaction. Upon characterization of the interaction of Hg(II) ion with TEM-1 β-lactamase ESI MS and 199mHg-PAC spectroscopic methods were applied. The results proved that Hg(II) could bind to the TEM-1 β-lactamase weakly. However, no specific Hg(II) adduct could be detected, suggesting that Hg(II) can be bound to one or more similar binding sites. Inspecting the available crystal structures, indeed more similar potential binding sites, composed of methionine side chains complemented with further weakly coordinating amino acid sidechains were identified. We have revisited the kinetics of the with TEM-1 β-lactamase promoted ampicillin hydrolysis. The hydrolytic process was described by the kinetic analysis of the set of catalytic progress curves recorded at multiple wavelengths, including various concentrations (and concentration ratios) of the enzyme and substrate. These experime

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