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

• Published in: Biochimica et biophysica acta 2011-10, Vol.1814 (10), p.1219-1225
• Main Authors: Tanaka, Kumiko, Matsumoto, Tamotsu, Suzuki, Tomohiko
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence - physiology; Amino Acid Substitution - physiology; Animals; Arenicola; Arenicola (polychaete); Arenicola brasiliensis; Binding Sites - genetics; Glycocyamine kinase; Mitochondrial Proteins - chemistry; Mitochondrial Proteins - genetics; Mitochondrial Proteins - metabolism; Models, Biological; Molecular Sequence Data; Mutagenesis, Site-Directed; Phosphotransferases (Nitrogenous Group Acceptor) - chemistry; Phosphotransferases (Nitrogenous Group Acceptor) - genetics; Phosphotransferases (Nitrogenous Group Acceptor) - metabolism; Polychaeta - enzymology; Polychaeta - genetics; Polychaeta - metabolism; Protein Binding - genetics; Protein Binding - physiology; Protein Structure, Tertiary - genetics; Sequence Analysis, Protein; Sequence Homology, Amino Acid; Substrate inhibition; Taurine - analogs & derivatives; Taurine - metabolism; Taurocyamine kinase
• ISSN: 1570-9639; 0006-3002; 1878-1454
• DOI: 10.1016/j.bbapap.2011.06.003

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 K m and V max (app K m and app V max, respectively) of the mutant forms for the substrates taurocyamine and glycocyamine. The app K m 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. ► 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.