The two-domain taurocyamine kinase (TK) from Paragonimus westermani was suggested to have a unique substrate binding mechanism. We performed site-directed mutagenesis on each domain of this TK and compared the kinetic parameters Km(Tc) and Vmax with that of the wild-type to determine putative amino acids involved in substrate recognition and binding. Replacement of Y84 on domain 1 and Y87 on domain 2 with R resulted in the loss of activity for the substrate taurocyamine. Y84E mutant has a dramatic decrease in affinity and activity for taurocyamine while Y87E has completely lost catalytic activity. Substituting H and I on the said positions also resulted in significant changes in activity. Mutation of the residues A59 on the GS region of domain 1 also caused significant decrease in affinity and activity while mutation on the equivalent position on domain 2 resulted in complete loss of activity.
Keywords: AK; CK; GK; LK; PK; Paragonimus westermani; Phosphagen kinase; Site-directed mutagenesis; TK; Taurocyamine kinase; Trematode; arginine kinase; creatine kinase; glycocyamine kinase; lombricine kinase; phosphagen kinase; taurocyamine kinase.
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