Pyrimidine salvage pathways are essential to all cells. They provide a balance of RNA synthesis with the biosynthetic pathway in pyrimidine prototrophs and supply all the pyrimidine requirements in auxotrophs. While the pyrimidine biosynthetic pathway is found in almost all organisms and is nearly identical throughout nature, the salvage pathway often differs from species to species, with aspects of salvage seen in every organism. Thus significant taxonomic value may be ascribed to the salvage pathway. The pyrimidine salvage pathways were studied in 55 microorganisms. Nine different salvage motifs, grouped I-IX, were identified in this study based on the presence of different combinations of the following enzymes: cytidine deaminase (Cdd), cytosine deaminase (Cod), uridine phosphorylase (Udp), uracil phosphoribosyltransferase (Upp), uridine hydrolase (Udh), nucleoside hydrolase (Nuh), uridine/cytidine kinase (Udk), 5'-nucleotidase and CMP kinase (Cmk).

<p>The HIV-1 virus encodes a protease essential for the processing of polyprotein precursors into mature viral proteins. This enzyme is a primary target for drug design against AIDS. Concurrent effects of inhibitors targeted to defined regions of the extended active site were investigated using Yonetani-Theorell kinetics to understand its complex specificity requirements. Kinetic data revealed that the simultaneous presence of two specific inhibitors may increase their binding affinity for the enzyme. A 100-fold enhancement in binding affinity was observed in certain instances. Results from this work showed a correlation between inhibitor synergism and substrate specificity thus implicating subsite interactions in enzyme catalysis.</p> <p>To facilitate the analysis of enzyme-inhibitor interactions an improved graphical method, the combination plot, was developed as an alternative to the Yonetani-Theorell plot. The method generates a single straight line rather than a family of lines which is the traditional approach in such kinetic studies. The slope of the plot, 1/α, quantitatively measures the extent and nature of interaction between two inhibitors on their target enzyme. The approach was easily extended to analyze, for the first time, the interaction between three competitive inhibitors on an enzyme. The combination approach potentially has broad applications for kinetic analysis.</p> <p>Combination plots were used in the discovery of gem-dialkyl succinic acid derivatives as a new class of unusually potent reversible inhibitors of carboxypeptidases A and B. 2-Ethyl-2-methylsuccinic acid binds to carboxypeptidases A and B with dissociation constants of 1.1 X 10ˉ⁷ M and 3.4 X 10ˉ⁶ M respectively. The low dissociation constants of the inhibitors for the zinc proteases can be attributed primarily to the gem-dialkyl groups which presumably make very important hydrophobic contacts within the active site. The inhibitors may also act as zinc ligands while possessing sufficient affinity for the carboxyl-recognition site in the enzymes.</p> / Doctor of Philosophy (PhD)

LD2668 .T4 1966 D656 / Master of Science

LD2668 .T4 1966 E38 / Master of Science

Call number: LD2668 .T4 1966 N631 / Master of Science

This research has been directed at the study of both the enzyme sulfite oxidase and molybdenum model chemistry. A modification of a previously published procedure has been used to purify sulfite oxidase in high yield which is well-suited for experiments requiring prosthetically intact enzyme and which is not contaminated with extraneous heme or with other redox active proteins. Laser flash photolysis was used to study the reaction of photoproduced 5-deazariboflavin, lumiflavin, and riboflavin semiquinone radicals with the redox centers of purified sulfite oxidase. Two distinctly different intramolecular electron transfer processes were observed between the molybdenum and heme sites of the enzyme, and these assignments were supported by flash photolysis studies of the cyanide-inactivated enzyme and the sulfite oxidase heme peptide. Microcoulometric experiments on sulfite oxidase have shown that the enzyme requires the addition of three electrons for complete subunit reduction. Midpoint potentials for the Mo(VI)/Mo(V), Mo(V)/Mo(IV), and Fe(III)/Fe(II) couples have been obtained under varied buffer conditions. The midpoint potentials obtained under High-pH and Low-pH conditions provided a means for reductively titrating the enzyme to the Mo(V) oxidation state for EXAFS studies. EXAFS of sulfite oxidase under High-pH and Low-pH conditions have provided the first example of a structural study of the three accessible oxidation states (Mo(VI), Mo(V), and Mo(IV)). A biologically relevant synthetic model for the formation of the Mo(V) Low-pH form of sulfite oxidase has been developed. The Mo(V) model compound closely resembles the minimum coordination environment for the Mo(V) Low-pH form of sulfite oxidase as determined by EXAFS. Using synchrotron radiation, molybdenum L-edge x-ray absorption spectra have been obtained for a variety of oxomolybdenum(V) compounds which serve as models for sulfite oxidase. An attempt has been made to correlate unique features of the molecules to the observed 2P → 4d electronic transitions.

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No description available.

published_or_final_version / Zoology / Master / Master of Philosophy

published_or_final_version / Biochemistry / Master / Master of Philosophy