Molecular Mechanism of Bifunctionality in an Ancestral Apicomplexan Dehydrogenase

DSpace Repository

Show simple item record

dc.contributor.advisor Theobald, Douglas en_US
dc.contributor.author Hood, Miriam
dc.date.accessioned 2018-05-14T17:48:43Z
dc.date.available 2018-05-14T17:48:43Z
dc.date.issued 2018
dc.identifier.uri http://hdl.handle.net/10192/35671
dc.description.abstract Malate dehydrogenases (MDH) and lactate dehydrogenases (LDH) are essential metabolic enzymes that share similar folds and mechanisms despite their strong substrate specificity. The unicellular eukaryotes of the Apicomplexa phylum, responsible for a variety of human diseases, have convergently evolved a unique and highly specific LDH from an MDH. The Apicomplexan LDH and MDH are an excellent model for the evolution of enzyme specificity due to the presence of a putative promiscuous intermediate in the evolutionary pathway. A six amino acid insertion into the active site conferred pyruvate activity by shifting the key catalytic residue from an arginine at position 102 in MDHs to a tryptophan at position 107f. However, the bifunctional intermediate contains both R102 and W107f. Though only one conformation is observed in x-ray crystallography data, I hypothesize that there are two active conformations of the intermediate. I used heteronuclear single quantum coherence nuclear magnetic resonance (HSQC NMR) to visualize a dimer of the promiscuous intermediate in the presence of each substrate. Loss of the tryptophan peak was seen in the presence of pyruvate at 25°C. I used intrinsic tryptophan fluorescence to monitor the catalytic tryptophan in the presence of each substrate. Pyruvate amplifies tryptophan fluorescence, but oxaloacetate demonstrates no consistent trend. Mutation of Trp107f to an alanine results in the loss of LDH activity but does not affect MDH activity. en_US
dc.description.sponsorship Brandeis University, Graduate School of Arts and Sciences en_US
dc.format.mimetype application/pdf en_US
dc.language English en_US
dc.language.iso eng en_US
dc.publisher Brandeis University en_US
dc.relation.ispartofseries Brandeis University Theses and Dissertations
dc.rights Copyright by Miriam Hood 2018 en_US
dc.subject Biochemisty en_US
dc.subject Enzymology en_US
dc.subject Protein Evolution en_US
dc.subject Specificity en_US
dc.subject Apicomplexan en_US
dc.subject Malate Dehydrogenase en_US
dc.subject Lactate Dehydrogenase en_US
dc.subject Ancestral Reconstruction en_US
dc.title Molecular Mechanism of Bifunctionality in an Ancestral Apicomplexan Dehydrogenase en_US
dc.type Thesis en_US
dc.contributor.department Department of Biochemistry en_US
dc.degree.name MS en_US
dc.degree.level Masters en_US
dc.degree.discipline Biochemistry en_US
dc.degree.grantor Brandeis University, Graduate School of Arts and Sciences en_US


Files in this item

This item appears in the following Collection(s)

Show simple item record

Search BIR


Browse

My Account