Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2006. / Includes bibliographical references (leaves 96-103). Also available in electronic version.

Thesis (Ph.D.) - University of Queensland, 2005. / Includes bibliography.

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 166-178).

Thesis (Ph.D.)--Cleveland State University, 2008. / Abstract. Title from PDF t.p. (viewed on Mar. 4, 2009). Includes bibliographical references. Available online via the OhioLINK ETD Center. Also available in print.

Quinolinate phosphoribosyltransferase (QPRTase, E.C. 2.4.2.19) is considered to be a unique enzyme in that it is thought to catalyse two distinct chemical reactions. Both the transfer of a phosphoribosyl group from 5-phosphoribosyl-1- pyrophosphate onto the nitrogen of quinolinic acid and the subsequent decarboxylation of the intermediate to form nicotinic acid mononucleotide are thought to be catalysed by the QPRTase system. Analogues of quinolinic acid were designed as potential inhibitors of QPRTase. These contain acidic groups at the 2- and 3- positions but are unable to decarboxylate. However, such compounds may be able to undergo the phosphoribosyl transfer reaction, potentially increasing their inhibitory potency. These compounds may be useful as "biological tools" allowing the neurological effects of an increase in quinolinic acid levels to be investigated. The compounds may show anti-fungal activity blocking the kynurenine pathway for NAD production. 2-Sulfonicotinic acid was synthesised by the oxidation of 2-mercaptonicotinic acid by either basic potassium permanganate, or iodine, with the structure was confirmed by X-ray crystallography. In biological testing the acid was shown to be neither an agonist nor antagonist of the NMDA receptor, or to be neurotoxic. A number of synthetic routes towards 2-phosphononicotinic acid, an alternative quinolinic acid analogue, were attempted though none were successful. These included orthometallation strategies and palladium coupling reactions to incorporate the phosphonic acid group at the 2- position. Nucleophilic addition routes, methods of building up the pyridine ring and including non-selective phosphonic acid addition were also examined. However, a related derivative, 2-(phosphonomethyl)nicotinic acid, was successfully synthesised. The non-enzymic decarboxylation of N-alkyl quinolinic acids was investigated, for comparison with the decarboxylation reaction catalysed by QPRTase. Both N- methyl and N-ethylquinolinic acid were synthesised, and the pH versus rate profiles measured. The rate maximum for both compounds was at pH 1.5, with the rate decreasing both above and below the maximum. N-Methylquinolinic acid was 10 times faster than quinolinic acid itself, demonstrating the effect of the nitrogen substituent. The N-ethyl derivative decarboxylated a further 1.5 times faster, showing the effect of increasing the size of the substituent. An Arrhenius plot was also carried out, giving an activation energy for the reaction of 153 kJ mol-1. Attempts to prepare the N-propyl derivative were unsuccessful, as decarboxylation occurred very readily to give N- propylnicotinic acid.

No description available.

Tyrosine phosphorylation is an important post-translational modification in cells that modulates key cellular pathways. Tyrosine phosphatases are the class of enzymes that remove this modification from proteins, yet we know relatively little about how they are regulated in various signaling contexts. Activity-based probes that successfully target active sites of tyrosine phosphatases and report on their activities can fill in this gap. We show the assessment of various thiol-reactive groups for their ability to target catalytic cysteine residues with specificity. Then we construct and screen a library of fragment-like compounds for their on-target and off-target reactivities. We also discuss theoretical considerations for screening covalent inhibitors for their kinetic parameters and show this using our experimental data. Lastly, we augment compounds selected from the library to enable click chemistry for reporter group attachment for use on the whole proteome, ultimately through mass spectrometry-based proteomics methods. We show enrichment of target proteins. These target-centric design efforts will yield new insights into the general development processes of activity-based probes or inhibitors.

Mammalian sperm are not able to fertilize immediately upon ejaculation; they become fertilization-competent after undergoing changes in the female reproductive tract collectively termed capacitation. Although it has been established that capacitation is associated with an increase in tyrosine phosphorylation, little is known about the role of this event in sperm function. In this work we used a combination of two dimensional gel electrophoresis and mass spectrometry to identify proteins that undergo tyrosine phosphorylation during capacitation. Some of the identified proteins are the mouse orthologues of human sperm proteins known to undergo tyrosine phosphorylation. Among them we identified VDAC, tubulin, PDH E1 β chain, glutathione S-transferase, NADH dehydrogenase (ubiquinone) Fe-S protein 6, acrosin binding protein precursor (sp32), proteasome subunit alpha type 6b and cytochrome b-c1 complex. In addition to previously described proteins, we identified two testis-specific aldolases as substrates for tyrosine phosphorylation. Genomic and EST analyses suggest that these aldolases are retroposons expressed exclusively in the testis, as has been reported elsewhere. Because of the importance of glycolysis for sperm function, we hypothesize that tyrosine phosphorylation of these proteins can play a role in the regulation of glycolysis during capacitation. However, neither the Km nor the Vmax of aldolase changed as a function of capacitation when its enzymatic activity was assayed in vitro, suggesting other levels of regulation for aldolase function. Looking upstream the kinase cascade, the identity of the kinase (s) that brings about the phosphorylation of the tyrosine residues remains to be elucidated. It has been suggested that the non receptor tyrosine kinase Src family is involved in the capacitation associated phosphorylation cascade. Using an immunological approach we show that the only Src family member present in mouse sperm extract is Src. The capacitation associated tyrosine phosphorylation is greatly reduced in the presence of Src specific inhibitors (SU6656 and SKI606) in vivo. As a means of control for the activity of Src inhibitors in our system, parallel experiments assaying the activity of PKA both in vivo and in vitro were realized. Surprisingly, Src inhibitors down regulates the phosphorylation of serine/threonine residues that correlate on earlier events in the capacitation, as assayed by western blot with PKA substrates antibody. However, in vitro kinase activity of PKA showed no effect of Src inhibitors in the phosphorylation of the PKA specific substrate, kemptide.

No / Recently two alternative mechanisms have been put forward for the inhibition of tyrosinase by 6R-l-erythro 5,6,7,8-tetrahydrobiopterin (6BH4). Initially allosteric uncompetitive inhibition was demonstrated due to 1:1 binding of 10¿6 M 6BH4 to a specific domain 28 amino acids away from the CuA active site of the enzyme. Alternatively it was then shown that 10¿3 M 6BH4 inhibit the reaction by the reduction of the product dopaquinone back to l-dopa. In the study presented herein we have used two structural analogues of 6BH4 (i.e., 6,7-(R,S)-dimethyl tetrahydrobiopterin and 6-(R,S)-tetrahydromonapterin) confirming classical uncompetitive inhibition due to specific binding of the pyrimidine ring of the pterin moiety to the regulatory domain on tyrosinase. Under these conditions there was no reduction of l-dopaquinone back to l-dopa by both cofactor analogues. Inhibition of tyrosinase by 6BH4 occurs in the concentration range of 10¿6 M after preactivation with l-tyrosine and this mechanism uncouples the enzyme reaction producing H2O2 from O2. Moreover, a direct oxidation of 6BH4 to 7,8-dihydrobiopterin by tyrosinase in the absence of the substrate l-tyrosine was demonstrated. The enzyme was activated by low concentrations of H2O2 (<0.3 × 10¿3 M), but deactivated at concentrations in the range 0.5¿5.0 × 10¿3 M. In summary, our results confirm a major role for 6BH4 in the regulation of human pigmentation.

The predicted protein product of open reading frame slr0328 from Synechocystis sp. PCC 6803, SynPTP, possesses significant amino acid sequence similarity with known low molecular weight protein tyrosine phosphatases (PTPs). To determine the gross functional properties of this hypothetical protein, open reading frame slr0328 was cloned, and its predicted protein product was expressed in E. coli. The recombinant protein, SynPTP, was purified by metal ion column chromatography. The catalytic activity of SynPTP was examined toward several exogenous protein substrates that had been phosphorylated on either tyrosine residues or serine residues. SynPTP exhibited phosphatase activity toward tyrosine phosphorylated protein substrates (Vmax toward phosphotyrosyl 32P-casein was 1.5 nmol/min/mg). However, no phosphatase activity was detected toward serine phosphorylated protein substrates. SynPTP displayed phosphohydrolase activity toward several organophosphoesters including para-nitrophenyl phosphate (p-NPP), beta-napthyl phosphate and phosphotyrosine but not toward alpha-napthyl phosphate, phosphoserine, or phosphothreonine. Kinetic analysis indicated that the Km (0.6 mM) and Vmax (3.2 mmole/min/mg) values for SynPTP toward pNPP are similar to those of other known bacterial low molecular weight PTPs. The protein phosphatase activity of SynPTP was inhibited by sodium orthovanadate, a known inhibitor for tyrosine phosphatases, but not by okadaic acid, an inhibitor for many serine/threonine phosphatases. Mutagenic alteration of the predicted catalytic cysteine, Cys7, to serine abolished enzyme activity. Several phosphotyrosine containing proteins were detected from the whole cell extracts of Synechocystis sp. PCC 6803 through immunoreactions using anti-phosphotyrosine antibody. SynPTP was observed to dephosphorylate three of these proteins in vitro. Two of these proteins were identified by peptide-mass fingerprinting analysis, as PsaD (photosystem I subunit II) and CpcD (phycocyanin rod linker protein). In addition, several phosphotyrosine proteins were detected from the soluble and membrane fractions of Synechocystis sp. PCC 6803 cell extracts by in vitro substrate trapping as potential endogenous substrates of SynPTP. Two of these proteins were identified as the alpha and beta subunits of phycocyanin. We therefore speculate that SynPTP might be involved in the regulation of photosynthesis in Synechocystis sp. PCC 6803. / Ph. D.