Thesis (M.S.)--University of Wisconsin--Madison, 1970. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (M.S.)--University of Wisconsin--Madison, 1970. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (M.S.)--University of Wisconsin--Madison, 1970. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

No description available.

Thesis (M.S.)--University of Wisconsin--Madison, 1970. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1969. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliography.

Thesis (Ph.D.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / The enzyme, deoxyribophosphoaldolase, reversibly cleaves deoxyribose- 5-P to glyceraldehyde-3-P and acetaldehyde. Until this exposition, it was not known to exist in human erythrocytes. However, the enzyme was postulated to occur in erythrocytes in order to account for the synthesis of glyceraldehyde-3-P observed in ghosts when deoxynucleosides were metabolized. Therefore, initial experiments were designed to detect acetaldehyde as an intermediate of deoxynucleoside metabolism by ghosts. Indeed, it was found in incubation mixtures containing deoxyinosine or deoxyadenosine as substrate. Accumulation of acetaldehyde was found to depend upon substrate concentration and no acetaldehyde was detected in the absence of substrate or in the presence of inosine or ribose-5-P. Acetaldehyde was identified spectrophotometrically by its reaction with yeast alcohol dehydrogenase and colorimetrically by its reaction with buffered semicarbazide solution. Confirmation of its identity was obtained by isolation of the 2,4 dinitrophenylhydrazone derivative of acetaldehyde aerated from incubation extracts. A direct relationship between acetaldehyde production and triosephosphate production to deoxyribose-5-P utilization by hemolysates prepared from human erythrocytes was shown. Conclusive evidence for the existence of deoxyribophosphoaldolase was obtained by isolating the enzyme from human erythrocytes. Two procedures were developed to isolate the enzyme. The first involved the use of Sephadex G-100 and DEAE-cellulose columns. The second procedure proved more fruitful and employed ammonium sulfate fractionation followed by elution from calcium phosphate gels. It was found that 26% of the original enzyme activity was recovered and purification was approximately 3,000 fold. Several characteristic properties of partially purified and purified preparations of the enzyme were studied. It was found that: 1. Enzyme activity decayed rapidly upon storage. Magnesium ion, cysteine HCl, beta-mercaptoethanol or reduced glutathione increased enzyme stability. In addition, the sulfhydryl containing compounds were able to partially reactivate previously inactivated enzyme. 2. The molecular weight of deoxyribophosphoaldolase was estimated by Sephadex gel fractionation to be slightly greater than 68,000, the molecular weight of hemoglobin. Because of its low affinity for DEAE-cellulose and high affinity for carboxy methyl cellulose, the enzyme appeared to be a very basic protein. 3. The reaction catalyzed by deoxyribophosphoaldolase favored cleavage of deoxyribose-5-P. At equilibrium, 60% of deoxyribose-5-P was converted to products. When acetaldehyde and glyceraldehyde-3-P were used as substrates, 40% of each was converted to deoxypentose product. 4. The enzyme showed a high specificity for each of its three substrates. No reaction of the enzyme occurred with ethyl alcohol, pyruvate, ribose-5-P, deoxyribose, lactate and dihydroxyacetone phosphate. 5. The enzyme reacted optimally at pH 6.5. 6. Deoxyribophosphoaldolase was activated by several carboxylic acids. The degree of activation was greater in the presence of citric acid than any dicarboxylic acid tested. An optimal activation of enzyme occurred when the concentration of citrate was varied between 3-15 umoles/ml. Citrate activation did not appear to reside in its ability to act as a chelator. Comparison of enzyme elution from Sephadex G-100 columns in the presence and absence of citrate suggested that citrate causes enzyme aggregation by a still unexplained mectanism. 7. The apparent Michaelis constants for deoxyribose-5-P cleavage were determined in the presence and absence of citrate and were found to be 6.4 x 10^-4 moles/liter and 24.0 x 10^-4 moles/liter respectively. The presence of deoxyribophosphoaldolase in human erythrocytes can clearly explain the production of triosephosphate and acetaldehyde from deoxynucleoside substrates and may, in fact, provide a major catabolic pathway for the deoxypentose moiety of these deoxynucleosides. The enzyme provides a simple mechanism for triosephosphate formation from deoxypentose and may be part of a pathway that converts deoxypentose phosphate to pentose phosphate. / 2999-01-01

The research summarized in this thesis focuses on directed evolution and enzyme mechanism studies of two aldolases: 2-deoxyribose-5-phosphate aldolase (DERA) and fructose-6-phosphate aldolase (FSA). Aldolases are nature’s own catalysts for one of the most fundamental reactions in organic chemistry: the formation of new carbon-carbon bonds. In biological systems, aldol formation and cleavage reactions play central roles in sugar metabolism. In organic synthesis, aldolases attract great attention as environmentally friendly alternative for the synthesis of polyhydroxylated compounds in stereocontrolled manner. However, naturally occurring aldolases can hardly be used directly in organic synthesis mainly due to their narrow substrate scopes, especially phosphate dependency on substrate level. Semi-rational directed evolution was used in order to investigate the possibility of expanding the substrate scope of both DERA and FSA and to understand more about the relationship between protein structure and catalytic properties. The first two projects focus on the directed evolution of DERA and studies of the enzyme mechanism. The directed evolution project aims to alter the acceptor substrate preference from phosphorylated aldehydes to aryl-substituted aldehydes. Effort has been made to develop screening methods and screen for variants with desired properties.  In the study of enzyme mechanism where enzyme steady state kinetic studies were combined with molecular dynamic simulations, we investigated the role of Ser238 and Ser239 in the phosphate binding site and the possible connection between enzyme dynamics and catalytic properties. The other two projects focus on the directed evolution of FSA and the development of a new screening assay facilitating screening for FSA variants with improved activity in catalyzing aldol reaction between phenylacetaldehyde and hydroxyacetone. The new assay is based on a coupled enzyme system using an engineered alcohol dehydrogenase, FucO DA1472, as reporting enzyme. The assay has been successfully used to identify a hit with 9-fold improvement in catalytic efficiency and to determine the steady state kinetic parameters of wild-type FSA as well as the mutants. The results from directed evolution illustrated the high degree malleability of FSA active site. This opens up possibilities to generate FSA variants which could utilize both aryl-substituted donor and acceptor substrates.

The carbonate radical anion CO3•- is a potent reactive oxygen species (ROS) produced in vivo through enzymatic one-electron oxidation of bicarbonate or, mostly, via the reaction of CO2 with peroxynitrite. Due to the vitally essential role of the carbon dioxide/bicarbonate buffer system in regulation of physiological pH, CO3•- is arguably one of the most important ROS in biological systems. So far, the studies of reactions of CO3•- with DNA have been focused on the pathways initiated by oxidation of guanines in DNA. In this study, low-molecular products of attack of CO3•- on the sugar-phosphate backbone in vitro were analyzed by reversed phase HPLC. The selectivity of damage in double-stranded DNA (dsDNA) was found to follow the same pattern C4′ > C1′ > C5′ for both CO3•- and the hydroxyl radical, though the relative contribution of the C1′ damage induced by CO3•- is substantially higher. In single-stranded DNA (ssDNA) oxidation at C1′ by CO3•- prevails over all other sugar damages. An approximately 2000-fold preference for 8-oxoguanine (8oxoG) formation over sugar damage found in our study identifies CO3•- primarily as a one-electron oxidant with fairly low reactivity toward the sugar-phosphate backbone.