title: Amino acid residues 62 and 193 play the key role in regulating the synergism of substrate binding in oyster arginine kinase.
abstract: The purpose of this study is to clarify the amino acid residues responsible for the synergism in substrate binding of arginine kinase (AK), a key enzyme in invertebrate energy metabolism. AKs contain a pair of highly conserved amino acids (D62 and R193) that form an ion pair, and replacement of these residues can cause a pronounced loss of activity. Interestingly, in the oyster Crassostrea AK, these residues are replaced by an N and a K, respectively. Despite this replacement, the enzyme retains high activity and moderate synergism in substrate binding (Kd/Km=2.3). We replaced the N62 by G or D and the K193 by G or R in Crassostrea AK, and also constructed the double mutants of N62G/K193G and N62D/K193R. All of the mutants retained 50-90% of the wild-type activity. In N62G and N62D mutants, the Kmarg for arginine binding was comparable to that of wild-type enzyme, but the Kdarg was increased 2-5-fold, resulting in a strong synergism (Kd/Km=4.9-11.3). On the other hand, in K193G and K193R mutants, the Kmarg was increased 4-fold, and synergism was lost almost completely (Kd/Km=1.0-1.4). The N62G/K193G double mutant showed similar characteristics to the K193G and K193R mutants. Another double mutant, N62D/K193R, similar to the amino acid pair in the wild-type enzyme, had characteristics similar to those of the wild-type enzyme. These results indicate that the amino acid residues 62 and 193 play the key role in mediating the synergism in substrate binding of oyster arginine kinase.
authors.affiliation: Laboratory of Biochemistry, Faculty of Science, Kochi University, Kochi 780-8520, Japan.
authors.forename: Naka
authors.initials: N
authors.lastname: Fujimoto
authors.forename: Kumiko
authors.initials: K
authors.lastname: Tanaka
authors.forename: Tomohiko
authors.initials: T
authors.lastname: Suzuki
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001120:Arginine / Q000378:metabolism; D001122:Arginine Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D017124:Conserved Sequence / Q000235:genetics; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D010125:Ostreidae / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D000596:Amino Acids; D001120:Arginine; D001122:Arginine Kinase