Identification of amino acid residues responsible for taurocyamine binding in mitochondrial taurocyamine kinase from Arenicola brasiliensis

In order to investigate the residues associated with binding of the substrate taurocyamine in Arenicola mitochondrial taurocyamine kinase (TK), we performed Ala-scanning of the amino acid sequence HTKTV at positions 67–71 on the GS loop, and determined apparent Km and Vmax (appKm and appVmax, respectively) of the mutant forms for the substrates taurocyamine and glycocyamine. The appKm values for taurocyamine of the K69A, T70A and V71A mutants were significantly increased as compared with wild-type, suggesting that these residues are associated with taurocyamine binding. Of special interest is a property of V71A mutant: its catalytic efficiency for glycocyamine was twice that for taurocyamine, indicating that the V71A mutant acts like a glycocyamine kinase, rather than a TK. The role of the amino acid residue K95 of Arenicola MiTK was also examined. K95 was replaced with R, H, Y, I, A and E. K95R, K95H and K95I have a 3-fold higher affinity for taurocyamine, and activity was largely lost in K95E. On the other hand, the K95Y mutant showed a rather unique feature; namely, an increase in substrate concentration caused a decrease in initial velocity of the reaction (substrate inhibition). This is the first report on the key amino acid residues responsible for taurocyamine binding in mitochondrial TK.

Research highlights
► First identification of residues for substrate binding in mitochondrial TK.
► Through Ala scanning, K67, T70 and V71 were shown to be associated with it.
► V71A caused a dramatic change, shifting the enzyme from TK to behaving like a GK.
► Typical substrate inhibition was observed for the K95Y mutant.
► The above inhibition was abolished in the V71A/K95Y double mutant.