Thesis supervisor: Stefan Strack. Includes bibliographic references (p. 77-88).

Thesis (Ph. D.)--University of Lund, 1995. / Published dissertation.

Thesis (Ph. D.)--University of Lund, 1995. / Published dissertation.

Thesis (Ph.D)--Boston University / The purpose of the research was to investigate the action of prostatic acid phosphatase on a spectrum of physiologically significant phosphate esters under varying environmental conditions. The effects of varying pH, substrate concentration, and enzyme concentration, the influence of inhibitors, and the influence of time on enzymatic hydrolysis were studied with each substrate. The enzymes extracted from both normal and cancerous prostatic tissue were used in an attempt to discover any differences existing between enzymes. A survey of the literature was made. A historical review of the history of acid phosphatase, its distribution in animal tissues and the influence of experimental conditions on enzyme activity is included in the body of the dissertation. Little work has been done to investigate the action of acid phosphatases on physiologically significant substrates, and the comparison of normal and cancerous enzyme preparations from human tissues has attracted the attention of only several investigators. [TRUNCATED]

Reversible phosphorylation of proteins on serine, threonine and tyrosine residues, is now widely accepted to be the principal mechanism for the control of intracellular events in eukaryotic and prokaryotic cells. The nodularins are known to be potent inhibitors of serine/threonine protein phosphatases, PPlc and PP2Ac, with sub-nanomolar inhibition constants. They have been shown to form covalent adducts with the enzymes and are known to be potent hepatotoxins and liver promoters. Nodularin has the general structure: cyclo [(R)-eryphro-beta-methyl-iso-Asp-(S)-X-Adda-(R)-iso- Glu-N-methyldehydrobutyric acid)], where (S)-X is a variable S-amino acid and Adda is the unique beta-amino acid, (2S,3S,8S,9S)-3-amiao-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6- dienoic acid. In order to investigate the mode of inhibition and also to probe the active-site binding interactions, we decided to synthesis new analogues of nodularin. Specific inhibitors for either PP1 or PP2A are not presently available, but would be useful biochemical tools in delineating the individual physiological roles of these enzymes. We decided to syntliesise, the potential inhibitor cyclo [betaAla-(2R)-Glu-alpha-OMe-gamma-Pro-(2R)-Asp-alpha-OMe-gamma-(25)-Phe], a stripped-down nodularin macrocycle, and also an analogue which is suitable for synthetic elaboration at the "Adda position". Using solution phase peptide synthesis (LPPS), four such nodularin analogues (both (25)- and (2R)-proline) were synthesised in seventeen steps. The cyclisation between the Phe and Asp residues were carried out using DIPEA under conditions of high dilution. NMR studies (TOCSY, ROESY) have elucidated the three dimensional structures which have been shown to be similar to the natural product, nodularin. A shorter synthesis of these nodularin analogues was developed using solid phase peptide synthesis (SPPS). Two solid phase synthesise of the nodularin macrocycles, cyclo-[betaAla-(2R)- Glu-alpha-OMe-gamma-Pro-(2R)-Asp-beta-(25)-Phe]; one in which Fmoc-(2S)-Phe-betaAla-(2R)-Glu-alpha-OMe-gamma-Pro-(2R)-Asp(alphaO-Wang Resin)-beta-OAllyl is deprotected and then cyclised on the resin prior to cyclisation were found to be successful. Even though the resin-bound synthesis gave low yields for the cyclisation step, compared to the situation in solution, it offered advantages in the construction of the linear isopentapeptide precursor. Initial studies have shown that the nodualrin analogues 130 and 131 are moderate inhibitors (IC50 2.8 mmol) of PP1 when tested using the malachite green system. Studies towards the synthesis of incorporating more suitable Adda functionalities, and the development of a radiolabelled protein phosphatase assay are currently being investigated within the group.

The effects of gamma radiation and restriction endonuclease (RE) induced DNA double strand breaks (dsb) upon the mutation frequency and the surviving fraction of three Chinese hamster cell lines V79-4, CHO-K1 and an X-ray sensitive dsb repair deficient cell line xrs-5 were studied. The X-ray sensitive xrs-5 cell line was shown to be more sensitive to both the lethal and the mutagenic effects of gamma radiation having a substantially lower surviving fraction and a higher thymidine kinase (tk) mutation frequency per unit dose than the parental CHO-K1 cells. The frequency of induced hprt- mutations in the V79-4 cell line was comparable to the induced frequency of tk mutations in the CHO-K1 cells. The effect of blunt- and cohesive- ended dsb upon the surviving fraction and the induced mutation frequency was studied by porating different Chinese hamster cell lines (CHO-K1, V79-4 and xrs-5) with RE using Streptolysin O (SLO). The surviving fraction of the different cell lines was reduced with increasing concentrations of Pvu II. Increases in the concentration of Pvu II produced increases in the frequency of hypoxyanthine guanine phosphoribosyl transferase (hprt) mutations in the V79-4 cells and tk mutations in the CHO-K1 and xrs-5 cells. However, the xrs-5 cells were shown to be hypomutable to Pvu II compared with the parental CHO-K1 cells. EcoR1 was ineffective at inducing tk mutations in the CHO-Kl cells but was as effective as Pvu II at inducing hprt mutations in the V79-4 cells. None of the spontaneously induced V79-4 hprt- mutant cells were shown to have observable molecular deletions when analysed by PCR deletion screening. One third of the radiation induced hprt - mutants were shown to be deletions. However, too few mutant cells were analysed for any non-random distribution of deletions to be observed. Half of the hprt- mutants induced by SLO poration alone were shown to be due to deletions of oi\e or more exons. The distribution of the DNA deletions in SLO hprt- mutations appeared to be nonrandom. The PCR amplification products of exons 7&8 were more frequently lost than any of the other exons in the hprt gene. It has been suggested that the SLO provided by the manufacturer used to porate the cells was contaminated with small amounts of endonucleases and exonucleases. The Pvu II induced deletions also appeared to be non-randomly distributed. The PCR amplification products of exon 2 were more frequently absent than any of the other exon products. Sequence data from the EMBL library indicated that Pvu II had a restriction site adjacent to the exon 2 nucleotide sequence of the Chinese hamster hprt gene but not in or bordering the other exons. This provides evidence that the blunt-ended dsb plays a role in the production of mutations. Mutation studies indicated that there is only one active copy of the autosomally located thymidine kinase gene in the CHO-K1 cells and their daughter cell lines (Singh and Bryant, 1991). However, whether the other homologous copy has been inactivated or deleted is not known. Experiments attempting to locate the thymidine kinase gene(s) were performed using FISH and a mouse tk cDNA probe. However, these attempts were unsuccessful.

Enzymic phosphate monoester hydrolysis by inositol monophosphatase from bovine brain (EC 3.1.3.25) occurs via the direct displacement of phosphate by water rather than by a two step mechanism involving a phosphorylated enzyme intermediate. The catalytic process is believed to involve two Mg2+ ions, one of which is buried and acts as a Lewis acid and phosphate coordination site. The second metal ion appears to coordinate to the alkyl phosphate bridging oxygen, the 'catalytic' hydroxyl group (C-60H of D-Ins 1-P) and to the nucleophile, water or hydroxide. Mechanistic differences have been identified between the hydrolysis of inositol phosphate and nucleoside 2'-monophosphate substrates in that although phosphate-oxygen ligand exchange with the solvent Is facile in the presence of inositol, no such exchange occurs in the presence of adenosine. The minimum structural requirements of a substrate have been demonstrated via synthesis of ethane 1,2-diol monophosphate which shows enzyme activity (Vmax ca. 12% that of Vmax for Ins 1-P, Km = 0.7 mM and Ki = 1.0 mM). Elaboration of the free hydroxyl group to produce (S,R)-, (S)- and (R)-pentane 1,2,5-triol 2-phosphate gave inhibitors of contrasting potency ((S)-pentane 1,2,5-triol 2-phosphate Ki = 0.12 mM, (R)-pentane 1,2,5-triol 2-phosphate, Ki = 3.8 mM) as expected from the structural requirements for hydrolysis to occur. The proposed mechanism of adenosine 2'-monophosphate hydrolysis involving the N3-atom of the adenine moiety has been discounted through spectroscopic analysis of enzymic incubations of new nucleoside substrates (Uridine 2'-monophosphate; Vmax 230% that of Vmax for 2'-AMP, Km = 4.0 mM and 5,6-dihydrouridine 2'-monophosphate; Vmax 70% that of Vmax for 2'-AMP, Km = 1.4 mM), which showed no intermediate phosphates, with only the substrate and final enzymic product (uridine) detected. Ethane 1,2-diol phosphate has been further elaborated to produce 2-methoxyethanol phosphate, diethylene glycol phosphate, pentane 1,5-diolphosphate, diethylene glycol cyclic phosphate and pentane 1,5-diol cyclic phosphate (Ki values range between 3 & 8 mM). Inhibition is attributed to interaction of the substrate with the second Mg2+ ion, and displacement of the catalytic water (hydroxide) molecule. The fact that the cyclic phosphate diesters are not hydrolysed to phosphate monoesters by the enzyme demonstrates that the attacking nucleophile is not positioned on the first (buried) Mg2+ ion. The mechanistic difference of nucleoside 2'-monophosphate hydrolyses is attributed to the ribofuranosyl oxygen acting as a surrogate for the catalytic hydroxyl group of inositol 1-phosphate. Modelling studies have shown that this results in 2'-AMP adopting an unfavourable conformation which is stabilised by the second (catalytic) Mg2+ ion. The absence of the phosphate moiety in adenosine prevents this conformation being achieved at the active site, accounting for the lack of inhibitory activity of adenosine, and the absence of phosphate- oxygen ligand exchange in the presence of adenosine. The proposed mechanism is consistent with all published kinetic data, and the substrate dependency of lithium inhibition.

Enzymic phosphate monoester hydrolysis by myo-inositol monophosphatase from bovine brain (EC.3.1.3.25) has been shown to occur via the direct displacement of phosphate by water rather than by a two step mechanism involving a phosphorylated enzyme intermediate. The catalytic process is believed to involve two magnesium ions, one of which is buried deep in the active-site cleft (Mg1) and co-ordinated the phosphate moiety of the substrate. The second metal ion (Mg2) is located closer to the opening of the active site and co-ordinates to the alkyl-phosphate bridging oxygen bond of the substrate. Detailed chemical and kinetic studies on the enzyme have defined many of the interactions of the natural substrate for the enzyme, inositol 1-phosphate, and has led to the proposal that Mg2, which is readily accessible from bulk solvent, should position and activate the nucleophilic water molecule through chelation. Independent X-ray crystallographic studies of protein-substrate complexes using inhibitory metal ions to prevent reaction has provided an almost identical picture of the active-site interactions. However, this study has placed the nucleophilic water molecule on Mg1. The two different sites for the nucleophile would give different stereochemical courses for phosphoryl transfer. A location on Mg1 would give inversion of configuration through an in-line displacement whereas attack by a water molecule chelated to Mg2 would give retention via a novel adjacent association and pseudorotation. In order to distinguish between these two mechanisms, the stereochemical course of the inositol monophosphatase reaction must be determined with respect to the phosphorus centre. A synthetic route has been developed to produce the chirally labelled substrate analogues of inositol 1-phosphate which are required to perform such a study. These compounds, (RP)- and (5P)-inositol l-[18O]phosphorothioate can now be produced with a high degree of isotopic enrichment. The absolute configuration at the phosphorus centre of each individual enantiomer has been determined indirectly using single crystal X-ray analysis. Preliminary work has also been carried out on the hydrolysis of these substrate analogues by inositol monophosphatase using [17O] water and the subsequent derivatisation of the chiral inorganic [16O, 17O, 18O] phosphorothioate product. Derivatisation of the inorganic phosphorothioate product is necessary in order to determine its absolute configuration at phosphorus. A comparison of this result with the known configuration of the substrate analogue which was processed, will allow the stereochemical course of the inositol monophosphatase reaction to be determined.

No description available.

No description available.