Mutagenesis of Phytophthora Sojae and Mitochondrial Taurocyamine Kinases to Determine Residues Important in Substrate Specificity

A good model for studying molecular evolution is the Phosphagen Kinase (PK), as it has a diverse family present in both unicellular and multicellular organisms. Through mutation, specialization, and divergence, this diverse PK family may have evolved a diverse set of substrate specializations, similar to what is seen in multicellular animals today. The evolutionary pathway of interest is that of the Glycocyamine Kinase (GK) from the Taurocyamine Kinase (TK). To investigate this pathway, the effect of making a one-residue mutation was observed to see the influence it would have on substrate binding and activity in PsTK and Ab MiTK enzymes. The mutations created to understand the enzymes’ activity were V71A, V71T and G26H in PsTK and V71T in Ab MiTK. The mutation in the V71T region of PsTK is suggested to be detrimental for the enzyme as it led to a dead enzyme that was unable to be transformed. The AbMiTK V71T mutant showed a better affinity and higher catalytic efficiency with glycocyamine, suggesting that this mutation was part of the pathway for the evolution of GKs. The binding affinity and the catalytic efficiency of the V71A mutant remained the same for glycocyamine but decreased for taurocyamine. This suggests that this was also part of the pathway for the evolution of GKs as the ability of the TK to use its substrate decreases by 10-fold. The G261H mutation leads to no observable enzyme activity, suggesting that the mutation is deleterious to the enzyme.