Mechanistic Characterization and Evolution of Adenylate Kinase

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dc.contributor.advisor Kern, Dorothee en_US
dc.contributor.author Murphy, Padraig Niall
dc.date.accessioned 2014-05-21T20:05:00Z
dc.date.available 2014-05-21T20:05:00Z
dc.date.issued 2014
dc.identifier.uri http://hdl.handle.net/10192/27231
dc.description.abstract In order to maintain homeotic nucleotide concentrations in the cell, the essential and ubiquitous adenylate kinase (ADK) catalyzes the reversible reaction of ATP and AMP to 2 ADPs. The mechanism, both conformational and chemical, was investigated extensively to characterize the role of the magnesium ion (Mg2+) cofactor and probe the transition state bonding scheme. Kinetic and NMR experiments performed with high concentrations of EDTA allowed for the determination of microscopic rate constants in the absence of a metal cofactor for the conformational step, lid opening, and the chemical step, phosphoryl transfer. The presence of a metal cofactor accelerated both lid opening and phosphoryl transfer by a factor of at least 103 and 104, respectively, with Mg2+ exhibiting the greatest chemical step enhancement on the order of 106. The lid opening and phosphoryl transfer rate constants for other divalent metals (Ca2+, Co2+, Mn2+… etc.) were also measured. When the rate constant of phosphoryl transfer was related to charge density of the metal ion, proximity to the charge density of Mg2+ was correlated to in an increase in rate constant. These results suggest that although lid opening is generally accelerated by the presence of a 2+ charge, the cofactor’s role in phosphoryl transfer is highly specialized for a cation of specific charge and size. The character of the transition state of phosphoryl transfer was examined through site-directed mutagenesis of highly conserved active site arginines in a thermophilic ADK. Mutations of catalytic arginins to lysines were used to interrupt residue-specific nucleophile and leaving group activation of the nucleotide substrates, contingent upon the direction of the reaction. The ratios of the forward and reverse rate constants for two mutants, R124K and R161K, were hypothesized to indicate the bonding scheme of the transition state, be it more pentavalent-like (tight) or metaphosphate-like (loose) in character. Because all of the observed rate constants were within an order of magnitude, on-enzyme equilibrium experiments were performed to establish relative populations of substrate-bound ADK at equilibrium. ADKs preference for the EADPADP state was maintained between the wild-type protein and the three arginine mutants, thereby demonstrating that nucleophile and leaving group activation was not specifically interrupted by our mutations. Therefore, it was impossible to discern the transition state bonding scheme from these experiments. Phylogenetic sequencing and ancestral reconstruction were carried out using three extant isoforms of ADK. By resurrecting ADK ancestors, we hoped to show that as the Earth cooled, entropic and enthalpic contributions to catalysis adjusted over time to account for the loss in available thermal energy. Through temperature dependences of two ADK ancestors, it was determined that the ancestors were not significantly active, likely due to the large phylogenetic distance between the starting protein sequences. For future investigations, an ancestral tree is being created from a single, bacterial genus, Bacillius. 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 Padraig Niall Murphy 2014 en_US
dc.title Mechanistic Characterization and Evolution of Adenylate Kinase 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


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