Examining Evolution of Substrate Specificity Using Taurocyamine Kinase

Phosphagen kinases are widely distributed across many different animals, protozoa, and bacterial species. They catalyze the reversible phosphorylation of the guanidino compounds using ATP. Phosphagen kinases mainly serve as a temporal buffering system in maintaining ATP homeostasis during periods of high energy demands. Eight different phosphagen kinases with a great conservation in structures and functions are distributed distinctly along the phylogenetic lines. While the monomeric AK is universally accepted as the ancestral PK, the evolutionary pathway from the ancestral AK to other dimeric PKs remains to be elucidated. The conventional pathway indicates that CKs evolving from the ancestral AK gave rise to other PKs. The other model counteracts with a new alternative pathway in which the dimeric AK evolving from the ancestral AK gave rise to TK that latter evolved into other PKs. The study is an attempt to explore the feasibility of the alternative pathway by more closely examining the hypothetical AK®TK and TK®GK pathways. The study used two TK mutants with a putative change of substrate specificity to arginine (H95Y mutant and G62D-H193R mutant) and two other TK mutants with a putative change of substrate specificity to glycocyamine (V71A and V71T). The attempts to induce H95Y and G62D-H193R mutants were unsuccessful. The kinetic examination of two other mutants (V71A and V71T) revealed an important role of the residue 71 in determining substrate specificity of PKs. However, the alternative pathway remains to be further investigated because of the variations in the kinetic results between the control and the experimental V71A mutant