The Influence of Identified Key Residues on Catalytic Activity and Substrate Specificity of Taurocyamine Kinases from Arenicola brasiliensis, An Enzyme in the Phosphagen Kinases Family

Tauracyamine kinases (TKs) are part of the phosphagen kinase family which act as an energy buffering mechanism in some invertebrates and unicellular organisms by catalyzing the transfer of phosphate from ATP to guanidino containing compounds, such as arginine, creatine and tauracycamine. TKs utilize tauroayamine as their primary substrate but many TKs exhibit promiscuity in substrate specificity and can also phosprylate glycocyamine or lumbricine. Multiple amino acid residues have been identified as key residues for substrate binding and/or specificity in the phosphagen kinases; specifically, amino acids residue 62-72 have been identified as key residues within the guanidino substrate (GS) loop that play an important role in substrate binding and preference. Previous work has shown that a single substitution in this loop can 'flip' substrate specificity from tauracyamine to glycocyamine although with a significant drop in activity. Using site-directed mutagenesis we have created additional substitutions in the GS loop to see if they can improve overall activity for the enzyme while still maintaining a preference for glycocyamine. We will be describing our kinetic analysis (KM , kcat , and kcat /KM ) of each of the variants and the wildtype TKs to help determine whether the residues mutated are critical in the determining the enzyme's preference and overall activity with the two substrates, glycocyamine and tauracyamine. This may lead to insight into how different substrate preferences may have evolved.