A mating between Escherichia coli 4318 (thi-leu-Las-Hfr) and E. coli A-1 (Met-Las+ F-) resulted in the formation of phototrophic recombinantshaving L-asparaginase activities at three distinct levels. The physiology of L-asparaginase synthesis in these recombinants is described.

A procedure was developed for the rapid isolation of human phosphoglucose isomerase by substrate-induced elution from cellulose phosphate. The high degree of selectivity of the elution provided homogenous enzyme from erythrocytes after a purification of approximately 30,000-fold with a recovery of approximately 70%. The enzyme was also isolated from other human tissues by a similar procedure.

No description available.

No description available.

Choline is universal among plants as phosphatidylcholine and in many plants it serves as a precursor for the compatible osmolytes glycine betaine and choline-𝘖-sulphate. In spinach, choline is synthesized by the sequential 𝘕-methylation of phosphoethanolamine (PEA) → phosphomethylethanolamine (PMEA) → phosphodimethylethanolamine (PDEA) → phosphocholine (PCho) and PCho is hydrolyzed to choline. The objective of this biochemical survey was to determine whether the activities of enzyme(s) converting PEA to PCho could be found in leaves of diverse plants and, if so, if common regulatory properties could be identified. Leaf tissue was harvested from 14 diverse dicot plants. Enzyme activities for the three sequential 𝘕-methylations were quantified in vitro and for representative assays using PEA as substrate, reaction products were identified by thin layer chromatography. Extracts of all plants tested could metabolize PEA to PMEA, the rate of conversion varied from 0.04 to 25 nmol · min⁻¹ · g⁻¹ Fresh wt for soybean and cotton, respectively. In vitro PMEA → PDEA and PDEA → PCho rates varied between the plant species tested. Both steps were highest in sugar beet (22 and 24 nmol · min⁻¹ · g⁻¹ Fresh wt for · PMEA → PDEA and PDEA → PCho, respectively) to below detection limits for soybean (<0.03 nmol· min⁻¹ · g⁻¹ Fresh wt). Upon dark exposure, PEA → PMEA reaction rates were reduced from the light period levels but not in all cases was the activity reduced beyond the level that could be attributed to a general loss in plant vigour. The reduction in 𝘕-methylation capacity was most pronounced in plants with the highest levels of PEA → PMEA activity, with reduction greatest in sugar beet > amaranth > spinach. Thus PEA → PMEA is catalyzed by a light-regulated enzyme in many but apparently not all dicot plants. Datko and Mudd (1988a) have proposed that PEA → PMEA conversion is ubiquitous among plants and the enzyme responsible catalyzes a committing step for PCho synthesis. PEAMeT activity was found in leaf extracts of 10 additional dicot species. The level of in vitro activity and light regulation is not equivalent among all plants examined. If in vitro rates faithfully reflect the in vivo capacity to synthesize choline, why the variability among plants with respect to their capacity to synthesize a universal metabolite? Alternate routes of PCho/phosphatidyl choline synthesis may explain these differences (Datko and Mudd, 1988a,b; Hanson and Rhodes, 1983; Hitz 𝘦𝘵 𝘢𝘭., 1981; Weretilnyk and Summers, 1992). Interestingly, the plants with the highest rates of PEA → PMEA activity, (cotton, sugar beet, amaranth, sunflower, spinach and statice) are all documented glycine betaine accumulating species. These plants may have higher rates of PEA methylation in order to meet requirements for osmolyte synthesis. Thus, it would be interesting to see if PEA metabolism to PCho is up-regulated in response to osmotic stress. / Thesis / Master of Science (MS)

Three 2à 2 factorial experiments (EXP) were conducted to investigate the underlying mechanism of corn quality and a supplemented cocktail enzyme of amylase, protease, and xylanase on broiler performance from 0 to 49 days of age. In each of the first two EXP, the four diets used consisted of (1) diet AR-/BR-; reduced dietary energy by increasing corn A matrix metabolizable energy (ME) 138 kcal/kg in EXP 1 or 125 kcal/kg ME with corn B in EXP 2; (2) diet AR+/BR+; AR-/BR- plus Avizyme 1502® (AZ); (3) diet AN-/BN-; normal energy diet; (4) diet AN+/BN+; AN-/BN- plus AZ. In EXP 3, four dietary treatments came from EXP 1 and 2 consisted of: AN-, AN+, BN-, and BN+. For each EXP, 1,440 male Ross 708 chicks were randomly assigned to one of the four dietary treatments (trts) with 9 replicates (reps)/trt and 40 chicks/ rep on day (d) 0. Body weight gain (BWG) and feed intake (FI) were determined on feed change days (d 14, 28, and 37) and on d 7 and 49. On d 28, subsets of birds were transferred to Petersime batteries to evaluate feed passage rate and nutrient retention. Digesta samples from gizzard, jejunum, and ileum as well as pancreatic tissue samples were collected for analyses of amylase, protease, and xylanase activities. Intestinal tissue samples were collected for determination of maltase, sucrase, and aminopeptidase N activities. Jejunal tissue on d 28 was also collected for total RNA isolation and a subsequent genome-wide microarray assay. On d 50, 54 birds per trt were processed to evaluate carcass yield. Interactions of ME and AZ were observed in both EXP 1 and 2. In EXP 1, interactions of ME and AZ on BWG were observed with higher values in birds fed AN- while lower in birds fed AN+, while opposite observations in EXP 2 with corn B diets. In both EXP 1 and 2, feed conversion ratio (FCR) in birds fed normal energy diets was better (P <0.05) as compared to birds fed reduced energy diets after d 28. Percent fatpad was increased (P <0.05) with dietary energy levels in EXP 1. Percent pectoral minor was increased (P <0.05) in birds fed AZ diets (4.80 vs. 4.62%). In EXP 3, interaction of dietary corn and AZ (P < 0.05) on BWG was noted during d 14 and 37. Birds fed corn B diets had better (P < 0.05) BWG and feed efficiency as compared to birds fed corn A diets until d 14. Nitrogen retention on d 30 was greater (P < 0.05) in birds fed AN- and BN+ as compared to birds fed AN+ and BN-. A 2.8% more apparent metabolizable energy with nitrogen correction (AMEn) in corn A diets was observed as compared to corn B diets. Corn A had higher amylase and xylanase activities as compared to corn B. Xylanase activity in jejunal and ileal digesta of birds fed corn A diets were higher (P < 0.05) as compared to that of corn B birds on d 7 and 49. Sucrase-isomaltase contributed 63, 80, and 74 % of the total maltase activity in duodenum, jejunum, and ileum. Sucrase activities in duodenum and jejunum were correlated (P < 0.05) with performance, whereas duodenal aminopeptidase N was negatively correlated (P < 0.05) with performance except period BWG. Pancreatic amylase and protease as well as gizzard protease and xylanase activities were correlated (P < 0.05) with performance. Number of jejunal genes regulated (P < 0.05) by corn variety was 77 as compared to those by enzyme supplementation in corn A diets (30 genes) and corn B diets (23 genes). Immune response and metabolism related genes were the most regulated genes in birds fed different corn diets without enzyme addition. In conclusion, enzyme supplementation improved broiler performance. Dietary formulation strategy using either reduced energy or normal energy in associated with enzyme supplementation should base on the feed ingredient quality. Corn quality may come from active components such as protease inhibitor and xylanase, and improved performance in birds fed high quality corn diets might also relate with minimal immune response and metabolic demand. / Ph. D.

Living cells manufacture and degrade thousands of chemical compounds in vivo. To do this cells rely on the activities of thousands of different protein catalysts distributed in aqueous interior compartments. Over the past several decades studies have shown that the thermodynamic and kinetic properties of most proteins, including enzymes, are different in vivo as compared to in vitro. Based on in vitro studies metabolic pathways have traditionally been thought to consist of intermediates randomly diffusing between soluble enzymes and are still portrayed as such in many biochemistry textbooks. A large number of metabolic pathways however are now known to exist as enzyme complexes due to molecular crowding effects in vivo. These differences have contributed to the controversy that surrounds explanations of how metabolic pathways are spatially organized and regulated in the living cell. The organization of enzymes in vivo is now thought to play a significant role in normal cellular physiology but evidence of this role, beyond intermediate channeling, is lacking. The long term goal of this work is to develop an experimental model and test the validity of theories concerning the spatial arrangement of enzymes in regulating metabolic pathways. The studies described in this dissertation have been focused on understanding how living cells organize metabolic pathways. I have examined some of the theoretical aspects of enzyme-enzyme interactions by modeling the complex formed by mitochondrial malate dehydrogenase and citrate synthase. These studies show that MDH and CS may bind in a specific orientation that facilitates the direct transfer of oxaloacetate from MDH to CS through a molecular channel. During these studies it was determined that A. thaliana does not encode stilbene synthase (STS), which catalyzes the first step in a pathway that competes with flavonoid biosynthesis in other plant species. Moreover, it was shown that flavonols are not required for pollen viability in A. thaliana as they are in maize and petunia. I also describe a novel method to clone fragments of DNA without ligase using the polymerase chain reaction (PCR). To establish an experimental model I have used a variety of techniques to analyze interactions between enzymes in the well-characterized flavonoid biosynthetic pathway in Arabidopsis thaliana. Evidence is presented that indicates that the first four enzymes in this pathway form a complex. Collectively this work suggests that the structural organization of enzymes into complexes is an important aspect of cellular metabolism and might directly impact the relative levels of specific compounds that are synthesized in vivo. / Ph. D.

Parkinson's disease (PD) is a chronic, progressive disorder of the central nervous system that affects approximately 1.5 million Americans. One of the principal pathological features of PD is dopamine deficiency in the substantia nigra of the brain. A key enzyme that has been associated with the neurodegeneration seen in PD is monoamine oxidase-B (MAO-B). Several inhibitors of this enzyme have resulted in neuroprotection in the mouse model of PD. One such compound is 7-nitroindazole (1). This thesis describes the synthesis and MAO activity of several indazolyl and benzimidazolyl prodrugs that are designed to release an enzyme inhibitor in the affected brain area. These studies have provided information regarding the nucleophilic aromatic substitutions of the ambident nucleophiles under consideration. We have also discovered a compound that releases the enzyme inhibitor upon bioactivation by MAO. These results as well as a MPTP mouse study with the aforementioned compound are detailed within. / Master of Science

Cyclophilins are ubiquitous enzymes that are involved in protein folding, signal transduction, viral proliferation, oncogenesis, and regulation of the immune system. Cyclophilin A is the prototype of the cyclophilin family. We use molecular dynamics to describe the catalytic mechanism of cyclophilin A in full atomistic detail by sampling critical points along the reaction coordinate, and use accelerated molecular dynamics to sample cis-trans interconversions. At these critical points, we analyze the conformational space sampled by the active site, flexibility of the enzyme backbone, and modulation of binding interactions.We use Kramer’s rate theory to determine how diffusion and free energy contribute to lowering the activation energy of prolyl isomerization. We also find preferential binding modes of several cyclophiln A inhibitors, and compare the conformational space sampled by inhibited cyclophilin A to the conformational space sampled during wild-type interactions. We also analyze the mechanism of the next family member cyclophilin B in order to probe differences in enzyme dynamics and intermolecular interactions that could possibly be exploited in isoform-specific drug design. Our results indicate that cyclophilin proceeds by a conformational selection binding mechanism that manipulates substrate sterics, electrostatic interactions, and multiple reaction timescales in order to speed up reaction rate. Conformational space sampled by cyclophilin when inhibited and when undergoing wild-type interactions share significant similarity. Cyclophilins A and B do have notable differences in enzyme dynamics, due to variation in intramolecular interactions that arise from variation in primary structures. This work demonstrates how computational methods can be used to clarify catalytic mechanisms.

abstract: Enzymes which regulate the metabolic reactions for sustaining all living things, are the engines of life. The discovery of molecules that are able to control enzyme activity is of great interest for therapeutics and the biocatalysis industry. Peptides are promising enzyme modulators due to their large chemical diversity and the existence of well-established methods for library synthesis. Microarrays represent a powerful tool for screening thousands of molecules, on a small chip, for candidates that interact with enzymes and modulate their functions. In this work, a method is presented for screening high-density arrays to discover peptides that bind and modulate enzyme activity. A viscous polyvinyl alcohol (PVA) solution was applied to array surfaces to limit the diffusion of product molecules released from enzymatic reactions, allowing the simultaneous measurement of enzyme activity and binding at each peptide feature. For proof of concept, it was possible to identify peptides that bound to horseradish peroxidase (HRP), alkaline phosphatase (APase) and â-galactosidase (â-Gal) and substantially alter their activities by comparing the peptide-enzyme binding levels and bound enzyme activity on microarrays. Several peptides, selected from microarrays, were able to inhibit â-Gal in solution, which demonstrates that behaviors selected from surfaces often transfer to solution. A mechanistic study of inhibition revealed that some of the selected peptides inhibited enzyme activity by binding to enzymes and inducing aggregation. PVA-coated peptide slides can be rapidly analyzed, given an appropriate enzyme assay, and they may also be assayed under various conditions (such as temperature, pH and solvent). I have developed a general method to discover molecules that modulate enzyme activity at desired conditions. As demonstrations, some peptides were able to promote the thermal stability of bound enzyme, which were selected by performing the microarray-based enzyme assay at high temperature. For broad applications, selected peptide ligands were used to immobilize enzymes on solid surfaces. Compared to conventional methods, enzymes immobilized on peptide-modified surfaces exhibited higher specific activities and stabilities. Peptide-modified surfaces may prove useful for immobilizing enzymes on surfaces with optimized orientation, location and performance, which are of great interest to the biocatalysis industry. / Dissertation/Thesis / Ph.D. Chemistry 2010