Identifying the Structural Determinants of Negative Cooperativity in Phosphagen Kinases

Abstract

When an enzyme exists as a dimer, the two subunits of the dimer are able to cooperate in such a way where the binding of a substrate in one subunit affects the other subunit's ability to bind substrates. In the case of negative cooperativity, the binding of substrates in one subunit decreases the ability for the other subunit to bind substrates. Currently it is unknown what structural features within a specific dimer allow for negative cooperativity to occur. The enzyme family phosphagen kinases can be used as a model system to study negative cooperativity, as some phosphagen kinases possess negative cooperativity while others do not. In order to determine potential structural determinants of negative cooperativity, the protein sequences and structures of both negatively cooperative and non‐cooperative PKs were compared. The presence of a specific histidine residue in the N‐terminal was identified as a possible structural determinant of negative cooperativity in PKs. To examine whether this histidine residue is a genuine structural determinant of negative cooperativity, a series of mutants will be created and the cooperativity of each will be determined via Isothermal Titration Calorimetry. Successful discovery of the structural determinant(s) of negative cooperativity in phosphagen kinases will allow for a better understanding of how negative cooperativity functions as a whole.