Life Sciences Commons^™

Investigating The Main Protease (Mpro) Of Sars-Cov-2 As A Potential Drug Target, Valerie Giovina Pascetta

Honors Theses and Capstones

The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 19 (COVID-19) pandemic has claimed the lives of roughly 6.2 million people worldwide as of May 2022. The virus’s main protease (M^pro ) has been identified as an attractive drug target due to the critical role it plays in the viral life cycle. The roughly 34 kDa M^pro cleaves functional viral polypeptides out of two long polyproteins at conserved cut sites, allowing them to fulfill their role in processes like transcription and replication. Here, we have studied the enzymatic activity …

Direct Single Molecule Imaging Of Enhanced Diffusion For Enzymes And Enzyme-Conjugated Dna Origami, Mengqi Xu

Dissertations - ALL

Enzymes have been shown to diffuse faster in the presence of their substrates. Recently, we revealed new insights into this process of enhanced diffusion using single-particle tracking (SPT) with total internal reflection fluorescence (TIRF) microscopy. We found that the mobility of individual enzymes was enhanced three-fold in the presence of the substrate, and the motion remained Brownian. We showed that the relative increase in diffusion is independent of the total enzyme concentrations; and the oligomerization state of enzymes did not change during the catalytic turnover. These experiments ruled out the possibility that the enhanced enzyme diffusion was caused by the …

The Discovery And Analysis Of Peroxidase Enzyme In Pueraria Montana, Kathryn Briley

Senior Honors Theses

Peroxidase enzymes are used for a variety of industrial and biotechnological applications because of their ease of purification, broad range of chemical activities, and low cost of use. Identification of quality peroxidase sources that are convenient for enzymatic isolation and give way to high yields of product is a desirable pursuit in biochemical research. Kudzu is an excellent candidate for this pursuit as it displays high catalytic activity in screening assays and is found in abundance. This project seeks to determine the enzyme stability, optimal pH conditions, and possible novel chemical activities of peroxidase isolated from kudzu leaves. The methods …

The Effect Of Alcalase Concentration On The Proteins From The Shells Of Litopenaeus Setiferus (White Shrimp), Liam T. Quan

Journal of the South Carolina Academy of Science

Chitin is a naturally abundant polymer that also happens to be biodegradable. Chitin can be used in a variety of different products such as biodegradable plastics, papers, medical products, foods, and medical treatments. To extract chitin, shells must be demineralized and deproteinized. The goal of this experiment was to examine the effect of the protease Alcalase in the deproteinization of litopenaeus setiferus shells. The hypothesis was that if the concentration of Alcalase increased, then the absorbance of proteins in the spectrophotometer reading would increase. The null hypothesis was that if the concentration increased there would be no change in absorption. …

Deciphering The Catalytic Mechanism Of Human Manganese Superoxide Dismutase, Jahaun Azadmanesh

Theses & Dissertations

The livelihood of human cells is heavily dependent on the ability to modulate the presence of highly reactive oxygen-based molecules termed reactive oxygen species (ROS). In excess, ROS facilitate oxidative damage to the macromolecules of cellular life. SODs are the major family of antioxidant proteins that prevent the buildup of overwhelming amounts of ROS within cells. Sometimes dubbed the “first line of defense” against oxidative damage, SODs defend against the harmful accumulation of ROS by eliminating superoxide. Superoxide is a ROS itself that is also a precursor to much more harmful ROS molecules. MnSOD is the manganese containing form of …

Determining A Method For Expressing And Purifying Cytochrome P450 4v2: A Protein Involved In Bietti's Crystalline Dystrophy, Cody Lane Turner

MSU Graduate Theses

Within the Cytochrome P450 class of enzymes, there are a group known as the “orphan” cytochromes. The “orphan” classification comes from the poorly understood in vivo functionality and substrate specificity. Cytochrome P450 4V2 (CYP4V2) is one of these “orphans” and belongs to the CYP4 family. The CYP4 family is known for the omega oxidation of endogenous fatty acids. This family is most commonly found on chromosome 1 (CYP4ABXZ). CYP4V2 is unique in that its location is bound to chromosome 4 as discovered by Jiao in 2004. Mutations within the CYP4V2 gene have been associated with the …

Investigation Of Potentially Catalytic Residues Of Uba5 Through Mutagenesis, Purification, And Structural Characterization, Grant Bradley

Senior Honors Projects, 2020-current

Ubiquitin-fold modifier 1 (Ufm1) is a member of the Ubiquitin (Ub) family of proteins whose primary function is degradation of proteins through a sequential mechanism of chemical reactions. Though Ufm1’s specific roles are largely unknown, this family of proteins has shown to play a part in a wide variety of processes, including regulation of the cell cycle¹, secretory functions of cells^2,3, and blood clotting⁴. Ufm1’s mechanism of action proceeds with the aid of three enzymes: an E1, E2, and E3. Uba5 is the E1 activating enzyme that is specific to Ufm1, and its mechanism of …

Development And Application Of Chemical Tools To Identify Kinase-Substrate Interactions, Aparni Kithulgoda Gamage

Wayne State University Dissertations

Post translational modifications regulate a variety of biological processes inside the cell.Protein phosphorylation is one such PTM modification catalyzed by protein kinases, which aid to transfer a signal from one place to another inside the cell. However, irregularities in kinase-mediated signaling are often implicated in many diseases, making kinases effective drug targets. To understand kinase-related disease formation and to discover drugs to treat these diseases, it is crucial to have a clear understanding on kinase-mediated cell signaling networks. A current gap in the kinase biology field is a lack of tools to identify which kinase phosphorylates which protein substrate inside …

Structural And Functional Characterization Of Cazyme And Cazyme-Related Proteins From: Bacteroides Thetaiotaomicron And Porphyromonas Gingivalis: Two Abundant Colonizers Of The Human Microbiome, James Stevenson

Theses and Dissertations (Comprehensive)

The human body consists of approximately 30 trillion cells, while non-human microbes that reside on and within the body outnumber human somatic cells by a factor of 1.3 – 2.3. The interplay between our cells and those of the colonizing microorganisms affect physiology in a multitude of ways, both beneficial and detrimental. Microbes found in the oral cavity, such as the Red Complex member Porphyromonas gingivalis, are associated with pathology, namely periodontal diseases including gum deterioration, tooth decay, and loss of underlying alveolar bone. At the other end of the gastrointestinal tract, microbes such as Bacteroides thetaiotaomicron are found …

Uncovering New Mechanisms Of Cdc34 And Cullin-Ring Activity, Spencer Hill

UNLV Theses, Dissertations, Professional Papers, and Capstones

Ubiquitylation is a cellular regulatory system found in all eukaryotic cells, which has managed to find a role in most pathways imaginable. The system works fundamentally through the ligation of a small protein known as ubiquitin onto a substrate. Depending on the context of the ubiquitin ligation, the substrate can be directed towards a number of cellular fates, the best-studied being degradation of the substrate. While originally thought of as a signal for cellular disposal units to degrade aberrant proteins, we now know that ubiquitin plays a highly nuanced role in cellular epistasis, controlling everything from the cell cycle to …

Transition State Interactions In A Promiscuous Enzyme: Sulfate And Phosphate Monoester Hydrolysis By Pseudomonas Aeruginosa Arylsulfatase, Bert Van Loo, Ryan Berry, Usa Boonyuen, Mark F. Mohamed, Marko Golicnik, Alvan C. Hengge, Florian Hollfelder

Chemistry and Biochemistry Faculty Publications

Pseudomonas aeruginosa arylsulfatase (PAS) hydrolyses sulfate and, promiscuously, phosphate monoesters. Enzyme-catalyzed sulfate transfer is crucial to a wide variety of biological processes, but detailed studies of the mechanistic contributions to its catalysis are lacking. We present linear free energy relationships (LFERs) and kinetic isotope effects (KIEs) of PAS and active site mutants that suggest a key role for leaving group (LG) stabilization. In LFERs PAS^_WT has a much less negative Brønsted coefficient (ß_{leaving group} ^obs-Enz=-0.33) than the uncatalyzed reaction (ß_{leaving group} ^obs=-1.81). This situation is diminished when cationic active site groups are exchanged for alanine. …

The Distinctive Regulatory Mechanisms Of Bacterial Acetyl-Coa Carboxylase, Alexandra Leigh Evans

LSU Doctoral Dissertations

Metabolic Regulation is a complex system used to control cellular metabolism in response to conditions in the cell’s environment. For most enzymes, the cell can rely upon a minimal amount of regulation; however, critical enzymes, such as acetyl-CoA carboxylase, must be regulated at multiple levels. Acetyl-CoA carboxylase catalyzes the first committed step in fatty acid synthesis. In bacteria, acetyl-CoA carboxylase forms a complex of three subunits–biotin carboxylase, biotin carboxyl carrier protein, and carboxyltransferase–which catalyze the carboxylation of acetyl-CoA to form malonyl-CoA via two half-reactions. In the first half-reaction, biotin covalently linked to biotin carboxyl carrier protein is carboxylated by biotin …

The N-Terminal Methyltransferase Homologs Nrmt1 And Nrmt2 Exhibit Novel Regulation Of Activity Through Heterotrimer Formation., Jon David Faughn

Electronic Theses and Dissertations

Protein, DNA, and RNA methyltransferases have an ever-expanding list of novel substrates and catalytic activities. Even within families and between homologs, it is becoming clear the intricacies of methyltransferase specificity and regulation are far more diverse than originally thought. In addition to specific substrates and distinct methylation levels, methyltransferase activity can be altered through formation of complexes with close homologs. This work involves the N-terminal methyltransferase homologs NRMT1 and NRMT2. NRMT1 is a ubiquitously expressed distributive trimethylase. NRMT2 is a monomethylase expressed at low levels and in a tissue-specific manner. They are both nuclear methyltransferases with overlapping target consensus sequences …

Site-Directed Mutagenesis Of Malate Dehydrogenase: A Class Project, Bruce J. Heyen, Chesley Rowlett, Jon Zatorski, Ryan Burch, Emily Veach, Andy Gemmaka

Scholar Week 2016 - present

Malate dehydrogenase (MDH) is an important enzyme in an organism’s metabolic pathways. MDH is found in almost all living cells and catalyzes the conversion of malate to oxaloacetate which also involves nicotinamide dehydrogenase (NAD) as a coenzyme. A method to study how an enzyme operates is to alter one of its amino acids and compare the activity of the enzyme before and after the mutation. As a class project in Advanced Biochemistry during the spring semester of 2018, we are working as a team to propose and carry out a point-based mutation on MDH.

Secondary Functions And Novel Inhibitors Of Aminoacyl-Trna Synthetases, Patrick Wiencek

Graduate College Dissertations and Theses

The aminoacyl-tRNA synthetases are a family of enzymes involved in the process of translation, more specifically, ligating amino acids to their cognate tRNA molecules. Recent evidence suggests that aminoacyl-tRNA synthetases are capable of aminoacylating proteins, some of which are involved in the autophagy pathway. Here, we test the conditions under which E. coli and human threonyl-tRNA synthetases, as well as hisidyl-tRNA synthetase aminoacylate themselves. These reactions are ATP dependent, stimulated by Mg2+, and are inhibited by increasing cognate tRNA concentrations. These data represent the foundation for future aminoacylation experiments, specifically delving into the relationship between the autophagy pathway and the …

Relationship Between Structure And Function In Nickel Proteins And Enzymes, Carolyn Carr

Doctoral Dissertations

Nickel is a rarely used but biologically important metal that is utilized in all three domains of life. In nickel utilizing organisms there is a corresponding trafficking system specifically designed to capture nickel, deliver, and export excess nickel to prevent toxic effects. It is critical to understand the mechanisms by which organisms achieve metal selectivity to duplicate or disrupt this process for the benefit of human health and to further understanding of regulation mechanisms in biology. RcnR is a Ni(II) and Co(II) responsive transcriptional regulator in E. coli. The research reported in this dissertation focuses on the relationship between …

A Study On The Interactions Of Trehalose With Model Folate Compounds, Carolyn R Ware

Chancellor’s Honors Program Projects

No abstract provided.

Enzymatically Active Microspheres For Self-Propelled Colloidal Engines, Jungeun Park

Dissertations and Theses

Micro- and nano-motors have attracted numerous attentions from various scientific areas due to their potential applications. Most studies on self-propelled colloidal engines have exploited catalytic decomposition of hydrogen peroxide to drive the motor. Since the hydrogen peroxide is caustic, it is not suitable to use in biological applications, encouraging people to develop “greener” fuels. The aim of this research is to study a new transduction mechanism for self-propulsion not tied to hydrogen peroxide, and which can in particular be used with biological molecules as fuels. In this study, we focus on making particles with enzymatic activity which can effectively decompose …

Mutant Study Of Sinorhizobium Meliloti Proline Utilization A (Puta), Jacob E. Wilkinson, John J. Tanner, Donald F. Becker

UCARE Research Products

The purpose of this project is to purify and characterize the reaction kinetics of mutant versions the enzyme Proline Utilization A (PutA) in Sinorhizobium meliloti. The enzyme catalyzes the first step in proline metabolism. It has two active sites. The first is proline dehydrogenase (PRODH) which converts proline to pyrroline-5-carboxylate (P5C). The second is P5C dehydrogenase (P5CDH) which converts P5C to glutamate. Although many bacterial organisms have PutA, there are still significant interspecies variations, resulting in an entire family of PutA enzymes. The main difference is the length of the amino acid sequence. This affects the protein’s structure or …

Identifying Non-Classical Active Sites As A Tool For Enzyme Inhibition, Marisol Serrano

Open Access Theses & Dissertations

Chagas disease, caused by the parasite Trypanosoma cruzi, is an endemic life-threatening disease that affects mainly the heart. It remains the leading cause of heart failure in Latin American countries. Since current treatments against this parasite are highly toxic and somewhat ineffective, novel and more efficacious types of interventions are desired. Cruzain, identified as the major cathepsin for T. cruzi, plays a major role in the parasite's life cycle; making this enzyme very attractive for potential trypanocidal drugs discovery. The recombinant cruzain is synthesized as a zymogenic pro-protein (PCZN) which possesses a pro domain and a catalytic domain. In this …

Characterization Of Cytidylyltransferase Enzyme Activity Through High Performance Liquid Chromatography, James Brault

Theses and Dissertations

The cytidylyltransferases are a family of enzymes that utilize cytidine 5â?? triphosphate (CTP) to synthesize molecules that are precursors to membrane phospholipids. There are four well known enzymes: CTP: phosphoethanolamine cytidylyltransferase (ECT), CTP: glycerol-3-phosphate cytidylyltransferase (GCT), 2-C-methyl-D-erythritol-4-phosphate cytidylyltransferase synthetase (CMS), and CTP: phosphocholine cytidylyltransferase (CCT). Previously, a radioisotope tagging method was employed to study cytidylyltransferase catalysis. Using CCT as a model, a method utilizing high performance liquid chromatography (HPLC) was developed to replace the radioisotope scintillation technique. The development of this new HPLC method is cheaper, more efficient, and more accurate than the previously established method.

The targets of separation …

Expression, Localization, And Kinetic Characterization Of The Phospholipid Biosynthesis Enzyme Ctp: Phosphocholine Cytidylyltransferase From The Protozoan Parasite Leishmania Major, Justin Daniel Theodore Lange

Theses and Dissertations

The eukaryotic parasite Leishmania is the causative agent of the disease leishmaniasis. L. major is the most common of 21 species that causes visceral leishmaniasis in humans, and 30 that cause the same disease in other mammals. Visceral leishmaniasis causes fever, weight loss, and over a short amount of time, multiple organ failure, and has a 100% mortality rate within 2 years. This makes it the second largest parasitic killer in the world behind malaria. Over 90% of the worldâ??s cases of visceral leishmaniasis have been reported in underdeveloped countries of India, Bangladesh, Nepal, Sudan, Ethiopia and Brazil, with 500,000 …

Synthesis, Characterization And Mechanistic Studies Of Biomolecules@Mesomofs, Yao Chen

USF Tampa Graduate Theses and Dissertations

Encapsulation of biomolecules is of great interest to research advances related to biology, physiology, immunology, and biochemistry, as well as industrial and biomedical applications such as drug delivery, biocatalysis, biofuel, food and cosmetics. Encapsulation provides functional characteristics that are not fulfilled by free biomolecules and stabilizes the fragile biomolecules. In terms of biocatalysis, solid support can often enhance the stability of enzymes, as well as facilitate separation and recovery for reuse while maintaining activity and selectivity. Various kinds of materials have been used for encapsulation of biomolecules, among which, porous materials are an important group. Metal-organic frameworks (MOFs) have attracted …

Amino Acid Racemase Enzyme Assays, Atanas D. Radkov, Luke A. Moe

Plant and Soil Sciences Faculty Publications

Amino acid racemases are enzymes that invert the α-carbon stereochemistry of amino acids (AAs), interconverting amino acids between their L- and D-enantiomers in a reversible reaction. In bacteria, they are known to have catabolic physiological functions but are also involved in the synthesis of many D-AAs, including D-glutamate and D-alanine, which are necessary components of the peptidoglycan layer of the bacterial cell wall. As such, amino acid racemases represent significant targets for the development of bactericidal compounds. Amino acid racemases are also regarded by the biotechnological industry as important catalysts for the production of economically relevant D-AAs. Here, we provide …

Rescuing Acetylcholinesterase From Nerve Agent Inhibition: Protein Dynamics Driven Drug Discovery, Aiyana M. Emigh, Brian Bennion

STAR Program Research Presentations

Severe morbidity and mortality consequences result from irreversible inhibition of human acetylcholinesterase by organophosphates (OPs). Oxime-based reactivators are currently the only available treatments but lack efficacy in the central nervous system (CNS) where the most damage occurs. Computational docking and molecular dynamics (MD) simulations reveal complex structural barriers that may reduce oxime efficacy. These results may guide future drug designs of more effective countermeasures.

Inhibitory Effects Of Novel Immucillin Analogues On Borrelia Burgdorferi Bgp Nucleosidase, Christian Guerrero, Seth Eidemiller, Ken Cornell

IDeA Network for Biomedical Research Excellence (INBRE)

The pathogenic spirochaete Borrelia burgdorferi causes Lyme disease and is transmitted by deer ticks when they feed. Lyme disease is multisystemic—it adversely affects the heart, joints, and skin. Recent studies demonstrate that B. burgdorferi possesses three methylthioadenosine/Sadenosylhomocysteine (MTA/SAH) nucleosidases essential for the catabolic breakdown of both MTA and SAH. Both MTA and SAH are by-products of major pathways involving Sadenosylmethionine (SAM) and are kept at low micromolar concentrations due to their inhibitory activity.

This project examined the effect of transition state inhibitors on the surface binding Borrelia glycosaminoglycanbinding protein (Bgp) nucleosidase using recombinant Bgp and whole-cell B. burgdorferi activity assays. …

Understanding The Thermodynamics Of Enzyme-Antibiotic Interactions With Aminoglycoside Phosphotransferase-3’-Iiia, Michele K. Miller

Chancellor’s Honors Program Projects

No abstract provided.

Developing A Protocol For Purifying The Mala Enzyme In Bdellovibrio Bacteriovorus, John Jared Trecker

Summer Research

The sequenced genome of the gram-negative predatory bacterium Bdellovibrio bacteriovorus contains a gene that encodes for malA, a putative maltase. Given the bacterium's observed disuse of prey carbohydrates, the gene's presence is mysterious. That characterization of the enzyme and studies of its activity and specificity can be better carried out, it is necessary to obtain pure enzyme. Protein was collected from lysed cultures of Top10/pmalA E. coli. Attempted purification by ion-exchange chromatography with DEAE columns produced significantly purer protein; SP ion exchange columns were unsuccessful, as were heparin and hydroxyapatite affinity columns. Gel filtration chromatography should prove a useful method …

Investigation Of Biochemistry And Enzymology Of Acyl-Coenzyme A Synthetase, Yu Meng

All Dissertations

Acetyl-CoA, an essential metabolite at the junction of various anabolic and catabolic pathways, is generated both from the breakdown of carbohydrates, lipids, and amino acids, and from the activation of acetate. AMP-forming acetyl-CoA synthetase, a key enzyme for acetate activation into acetyl-CoA in all domains of life, is a member of the adenylate-forming enzyme superfamily. Although members in the superfamily have different biological functions, they all share the property of forming an acyl-adenylate intermediate. Here I describe my research on characterization of a medium-chain acyl-CoA synthetase from Methanosarcina acetivorans (Macs_Ma) and identification of key residues in acyl substrate …

Characterization Of The Substrate Specificity And Mechanism Of Protein Arginine Methyltransferase 1, Whitney Lyn Wooderchak

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Protein arginine methyltransferases (PRMTs) posttranslationally modify protein arginine residues. Type I PRMTs catalyze the formation of monomethylarginine (MMA) and asymmetric dimethylarginine (ADMA) via methyl group transfer from S-adenosyl methionine onto protein arginine residues. Type II PRMTs generate MMA and symmetric dimethylarginine. PRMT-methylation affects many biological processes. Although PRMTs are vital to normal development and function, PRMT-methylation is also linked to cardiovascular disease, stroke, multiple sclerosis, and cancer.

Thus far, nine human PRMT isoforms have been identified with orthologues present in yeast, plants, and fish. PRMT1 predominates, performing an estimated 85% of all protein arginine methylation in vivo. Yet, the substrate …