Browsing by Author "Manary, Brandon"
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Item Designing a model for the enzyme–substrate complex to investigate the detailed catalytic mechanism of lactoperoxidase by quantum chemical methods(2015) Manary, Brandon; Llano, JorgeLactoperoxidase (LPO) is a heme enzyme found in exocrine secretions such as milk, saliva and endodermal mucus. LPO catalyzes the formation of oxidizing oxoanions that act as natural antibiotics in those body fluids. LPO is a versatile enzyme that converts thiocyanate ions (SCN−) to hypothiocyanite ions (OSCN−), as well as halide ions (i.e., Cl−, Br− and I−) to hypohalite ions (i.e., ClO−, BrO− and IO−). By means of quantum-mechanical–molecular-mechanical (QM–MM) calculations, this investigation aims to find the sequence of bond-breaking and bond forming steps by which LPO converts the anions into the oxoanions in its active-site. In this particular study, we present a molecular model for the LPO active-site, which was generated from a detailed analysis and comparison of the available crystallographic structures of LPO deposited in the Protein Data Bank. We also propose a chemical model for the enzyme–substrate complex, which will be used as the starting structure in the QM–MM computation of the LPO catalytic mechanism.Item Intermolecular forces at play in the active site of lactoperoxidase(2016) Manary, Brandon; Llano, JorgeLactoperoxidase (LPO) is an enzyme that fights in the first line of defense against infection. LPO catalyzes the formation of toxic chemicals which indiscriminately kill foreign microbes and viruses caught in the mucous membranes of vulnerable body parts (namely, the eyes and upper airways). Because of its importance for the immune system, LPO is largely studied for potential applications in medical therapies. However, while much research has been done to determine the protein's structure and its efficacy against various pathogens, the chemical mechanism by which the enzyme's active-site transforms common ions into germ-killing agents is not known in detail. The aim of this project is to apply methods of computational chemistry and bioinformatics implemented in state-of-the-art software to elucidate the catalytic mechanism of LPO with common substrates found in the body fluids.Item Molecular modelling and kinase assay for CePNKP binding to DNA(2017) Manary, Brandon; Bernstein, Nina; Llano, JorgeComputational techniques of homology modelling, enzyme–substrate docking, and molecular dynamics were applied to elucidate the structure and substrate binding properties of the DNA repair enzyme polynucleotide kinase/phosphatase from the nematode C. elegans (CePNKP). PNKP is involved in the repair of DNA strand breaks, a form of DNA damage caused by reactive oxygen species, ionizing radiation and certain chemical mutagens. Mutations in human PNKP have been associated with the neurological disorders Microcephaly with Intractable Seizures (MCSZ) and Ataxia Oculomotor Apraxia 4 (AOA4). In addition, human PNKP has been identified as a potential drug target for the development of chemo- and radiosensitizing agents for cancer treatment. CePNKP is a useful model system for studying the human enzyme. The substrate preference for both human and C. elegans PNKP has been investigated, and found to be similar, with higher selectivity for recessed over blunt DNA ends. However, CePNKP exhibits a more exclusive preference for recessed DNA ends than human PNKP. To elucidate the reason for the unique substrate specificity of CePNKP, the structure of CePNKP in complex with its DNA substrate must be determined in atomistic detail by molecular modelling. The generated structural model is compared with our experimental results of kinase activity assays of wild-type CePNKP.