(499.14) Determining Whether Tightness of Substrate Binding in the Active Site of a Dimeric Taurocyamine Kinase Plays a Role in Inducing Negative Cooperativity

Poster Board Number: A216

Robby Beal (The College of Wooster), Mingyuan Li (The College of Wooster), Dean Fraga (The College of Wooster), Mark Snider (The College of Wooster)

Cooperative binding of ligands within multi-subunit proteins is a mechanism that has evolved to enhance a protein’s function. Driven by conformational changes upon ligand binding in one subunit, the resulting change in affinity at a neighboring subunit can be enhanced (positive) or reduced (negative) depending on the mechanism. The structural features, both on the substrate and within the active site, that determine the nature of cooperativity remain largely unknown, particularly within the phosphagen kinases (PKs). PKs belong to a widely distributed enzyme family important for energy homeostasis, with isoforms that exist as monomers and multimers, with some, but not all, of the homodimers that have been studied exhibiting negative cooperativity. Here we investigated the structural determinants that elicit negative cooperativity of transition state analogue (TSA; TK–MgADP–NO3-–taurocyamine) binding within the Arenicola Brasiliensis mitochondrial taurocyamine kinase (miTK), one member of the phosphagen kinases that catalyzes the interconversion of MgATP and MgADP using taurocyamine or phosphotaurocyamine as substrates. Isothermal titration of miTK with taurocyamine in the presence of MgADP and nitrate to form the quaternary TSA complex fits a two-site binding model in which the first site forms a tighter complex than the second site (i.e. negative cooperativity). MiTK also shows activity with glycocyamine, although with reduced efficiency and weaker binding affinity. To determine whether this weakly bound substrate also elicits cooperativity we attempted to measure the wild-type miTK’s affinity for glycocyamine in forming a TSAC.  Results showed that this complex does not form appreciably and therefore the presence of negative cooperativity could not be elucidated. To further examine whether the tightness of binding or a specific structural feature of the substrate triggers cooperativity, the V71A active site variant of miTK that has shown to have 10x greater catalytic efficiency with glycocyamine compared to the wild-type miTK, will be assessed for cooperative TSAC binding using taurocyamine and glycocyamine. Additional active site variants with more moderate changes in affinity for these substrates will also be examined for cooperativity to further discern if negative cooperativity can be induced at a certain catalytic efficiency threshold.

The College of Wooster