PrnB is a heme-containing enzyme, which catalyzes the ring rearrangement reaction of 7-chlorotryptophan to produce 3-(3-Chloro-2-nitrophenyl)pyrrole. This thesis describes the initial isolation and characterization of PrnB, the second enzyme associated with the pyrrolnitrin biosynthetic pathway in Burkholderia ambifaria. Additionally, alternative peroxidase reactivity was used to study how amino-acids bind to the substrate binding pocket of PrnB. The peroxidase activity of PrnB was measured using three different peroxidase activity assays at various pH values. The peroxidase data was compared to similar studies with the classic peroxidase, Horseradish peroxidase (HRP). Generally, PrnB showed weak peroxidase reactivity. However this weak reactivity was an experimental handhold, where tryptophan and other substrate binding events can be explored using classic inhibition steady-state kinetics. The rate of 2-aminophenol oxidation by PrnB was used as a model assay to monitor how molecules such as L-tryptophan, L-alanine, indole, L-phenylalanine, and L-tyrosine interact with the PrnB active site.

Human carbonic anhydrases are metalloenzymes that are involved in many physiological processes in the body, but also play an important role in the pathogenesis of numerous diseases. Under regular conditions, expression of carbonic anhydrase IX (CAIX) is very limited, unlike that of other 14 human carbonic anhydrase isozymes. But in hypoxic tumors this enzyme is highly overexpressed on the cell surface. For this reason, this enzyme represents a good target for therapy and diagnosis of tumors and thus various anti-CAIX monoclonal antibodies and specific inhibitors are being developed. In this work we investigated the possibility to use fluorescent polymer conjugate carrying a CAIX specific inhibitor for florescent labeling of tumor cells. Specific binding of polymer conjugate to different cell lines was investigated by flow cytometry and confocal microscopy. Binding properties of the polymer conjugate was compared to CAIX specific monoclonal antibody M75 and its single-chain fragment scFv M75. Ability of the polymer conjugate to inhibit CAIX enzyme activity was also investigated. For these experiments, recombinant protein CAII was prepared and purified, which was also used for protein crystallization. Tests of inhibitory activities allowed to identify novel inhibitors CAIX with better inhibitory...

Aldehyde oxidase catalysed oxidation of various quinolinium and related cations has been studied in vitro. Oxidation products were identified by comparison of their spectral and chromatographic characteristics with those of authentic compounds. The N-heterocyclic cations and quinolones used required synthesis. Incubation of N-methylquinolinium, N-methyl-7,8-benzoquinolinium and N-phenylquinolinium yielded the corresponding 2- and 4-quinolones simultaneously. The ratio of 2- to 4-quinolone formation was found to be species dependent; the proportion of 4-quinolone was greater with guinea pig enzyme than with rabbit enzyme. Incubation of N-methyl-4-methylquinolinium, N-methyl-4-phenylquinolinium and N-methylphenanthridinium produced the expected 2-quinolones. Cations substituted adjacent to the ring nitrogen, i. e. N-methyl-2- methylquinolinium, N-methyl-2-phenylquinolinium and N-phenyl-2-phenylquinolinium, were oxidised to the corresponding 4-quinolones. Kinetic constants were determined spectrophotometrically. The Km values obtained with rabbit enzyme ranged from 1.6 x 10-3 M for N-methylquinolinium to <10-5 M for N-phenyl-2-phenylquinolinium. Quaternary compounds were found to be better substrates than their non-quaternary counterparts, except for N-methylisoquinolinium and N-methylphenanthridinium. In general, guinea pig aldehyde oxidase was shown to have a greater affinity for N-heterocyclic cations than rabbit enzyme. The substrate binding site has been discussed in the light of the results outlined below. Oxidation of N-methyl-4-phenylquinolinium (to the 2-quinolone) was competitively inhibited by N-methyl-2-phenylquinolinium (which yields the 4-quinolone), indicating that both these cations interact at the same active site. The ratio of 2- to 4-quinolone production from N-methylquinolinium was constant under various conditions, including purification of the enzyme but changed at high pH or in the presence of N-methylphenanthridinium. Inhibition studies indicated that both quaternary and non-quaternary compounds act at the same site on the enzyme. Km and Vmax values for phthalazine, N-methyl-2-phenylquinolinium and N-methylquinolinium were determined over the pH range 5.4 to 10.2. In each case, results indicated that the enzyme has an ionisable group at the active site with a pK ca. 8. Aldehyde oxidase was shown to catalyse the dehydrogenation of the pseudobases 3,4-dihydro-4-hydroxy-3-methyl-2-quinazolinone and 3,4-dihydro- 4-hydroxy-3-methylquinazoline.

Aldehyde oxidase catalysed oxidation of various quinolinium and related cations has been studied in vitro. Oxidation products were identified by comparison of their spectral and chromatographic characteristics with those of authentic compounds. The N-heterocyclic cations and quinolones used required synthesis. Incubation of N-methylquinolinium, N-methyl-7,8-benzoquinolinium and N-phenylquinolinium yielded the corresponding 2- and 4-quinolones simultaneously. The ratio of 2- to 4-quinolone formation was found to be species dependent; the proportion of 4-quinolone was greater with guinea pig enzyme than with rabbit enzyme. Incubation of N-methyl-4-methylquinolinium, N-methyl-4-phenylquinolinium and N-methylphenanthridinium produced the expected 2-quinolones. Cations substituted adjacent to the ring nitrogen, i. e. N-methyl-2- methylquinolinium, N-methyl-2-phenylquinolinium and N-phenyl-2-phenylquinolinium, were oxidised to the corresponding 4-quinolones. Kinetic constants were determined spectrophotometrically. The Km values obtained with rabbit enzyme ranged from 1.6 x 10-3 M for N-methylquinolinium to <10-5 M for N-phenyl-2-phenylquinolinium. Quaternary compounds were found to be better substrates than their non-quaternary counterparts, except for N-methylisoquinolinium and N-methylphenanthridinium. In general, guinea pig aldehyde oxidase was shown to have a greater affinity for N-heterocyclic cations than rabbit enzyme. The substrate binding site has been discussed in the light of the results outlined below. Oxidation of N-methyl-4-phenylquinolinium (to the 2-quinolone) was competitively inhibited by N-methyl-2-phenylquinolinium (which yields the 4-quinolone), indicating that both these cations interact at the same active site. The ratio of 2- to 4-quinolone production from N-methylquinolinium was constant under various conditions, including purification of the enzyme but changed at high pH or in the presence of N-methylphenanthridinium. Inhibition studies indicated that both quaternary and non-quaternary compounds act at the same site on the enzyme. Km and Vmax values for phthalazine, N-methyl-2-phenylquinolinium and N-methylquinolinium were determined over the pH range 5.4 to 10.2. In each case, results indicated that the enzyme has an ionisable group at the active site with a pK ca. 8. Aldehyde oxidase was shown to catalyse the dehydrogenation of the pseudobases 3,4-dihydro-4-hydroxy-3-methyl-2-quinazolinone and 3,4-dihydro- 4-hydroxy-3-methylquinazoline.

Bacteria have exhibited a remarkable capacity to become resistant to commonly used antibacterial compounds obliging the researchers to find new target to kill them. Peptidoglycan, a polymer which is completely specific to the bacterial world and the enzymes, is an ideal target. Peptidoglycan is formed by linear glycan chains composed of alternating N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) residues cross-linked by short peptides. Among the multitude of enzymes of degradation of the peptidoglycane, the N-acetylmuramyl-L-alanine amidase have the capacity to break the bond between the peptide and the lactyl grouping of MurNAc wearing this peptide. The subject of this work was to study AmiD, a N-acetylmuramyl-L-alanine amidase from E.coli. For that purpose, two carbohydrates 1,6-anhMurNAc with a protective group at the fourth position have been synthesised on gram scale in seven steps. After that, we have realized a structural study at the level of the peptide chain by miming the structure of the peptidoglycane. First, different amide compounds have been prepared. In a second time, we have synthesised carbohydrate compounds with one, two and three amino acids in the peptide chain. Finally, two carbohydrates with a triazole in the peptide chain have been prepared by click chemistry from a synthesised azoture precursor. All compounds have been synthesised with a chromatic group at the end of the peptide chain in order to facilitated the HPLC detection of the residue after hydrolysis by AmiD. Substrates studies, inhibition studies and kinetic studies have been realised with these carbohydrates. This work present also the first results of the synthesis of 1,6-anhydro-4-fluoroMurNAc, a possible inhibitor of bacterial growth. If the peptide chain contains a minimum of dipeptide residue (L-Ala--D-Glu), our results pointed that AmiD is able to cleave the amide bond between the lactyl group of the MurNAc and the -amino group of L-Ala. In the presence of a tripeptide chain (L-Ala--D-Glu-L-Lys) higher hydrolysis rates have been observed. Furthermore, the m-A2pm found in the natural substrate of AmiD can be replaced by L-Lys which facilitates the synthesis of the MurNAc derivatives.

The glyoxalase system, which has been known since 1913, is widely distributed in nature. The system consists of two enzymes, glyoxalase I and glyoxalase II. Methylglyoxal is very unstable and undergoes oxidation and polymerization reactions. One of the purposes of this study was to find a simple, convenient and reproducible method of methylglyoxal preparation. Another objective was the purification of both glyoxalase enzymes employing affinity chromatography as a major step. The purified enzymes were to be characterized by chemical, physical and kinetic properties as an approach to the understanding of the biological function of the system.

Quinolinate phosphoribosyltransferase (QPRTase, EC 2.4.2.19) is an intriguing enzyme which appears to catalyse two distinct chemical reactions; transfer of a phosphoribosyl moiety from 5-phosphoribosyl-1-pyrophosphate to the nitrogen of quinolinic acid and decarboxylation at the 2-position to give nicotinic acid mononucleotide. The chemical mechanism of QPRTase is not fully understood. In particular, enzymatic involvement in the decarboxylation step is yet to be conclusively proven. QPRTase is neurologically important as it degrades the potent neurotoxin, quinolinic acid, implicated in diseases such as Huntington’s disease and AIDS related dementia. Due to its neurological importance and unusual chemistry the mechanism of QPRTase is important. Described here is a mechanistic study on human brain QPRTase. Human brain QPRTase was successfully expressed in E. coli BL21 (DE3) from the pEHISTEV-QPRTase construct and the protein was efficiently purified by nickel affinity chromatography. The crystal structure was solved using multiwavelength methods to a resolution of 1.9 Å. Human brain QPRTase was found to adopt an energetically stable hexameric arrangement. The enzyme was also found to exist as a hexamer during gel filtration under physiological conditions. Kinetic studies allowed the measurement of the kinetic parameters for quinolinic acid. The data gave a Km of 13.4 ± 1.0 μM and a Vmax of 0.92 ± 0.01 μM min-1. There was no evidence for cooperative binding of quinolinic acid to the six subunits of the QPRTase hexamer. The enzyme showed maximum activity at approximately pH 6. The active site of human brain QPRTase is a deep pocket with a highly positive electrostatic surface composed of three arginine residues, two lysine residues and one histidine residue. Mutation of these residues resulted in either complete loss or significant reduction in enzymatic activity showing they are important for binding and/or catalysis. A possible mechanism involving QPRTase in the decarboxylation of quinolinic acid mononucleotide was proposed. A series of quinolinic acid analogues were synthesised and tested as inhibitors of QPRTase. The inhibition studies highlighted some key interactions in the active site.