Cooperativity in the two-domain arginine kinase from the sea anemone Anthopleura japonicus. II. Evidence from site-directed mutagenesis studies.
The arginine kinase (AK) from the sea anemone Anthopleura japonicus has an unusual two-domain structure (contiguous dimer; denoted by D1-D2). In a previous report, we suggested cooperativity in the contiguous dimer, which may be a result of domain-domain interactions, using MBP-fused enzymes. To further understand this observation, we inserted six-Lys residues into the linker region of the two-domain AK (D1-K6-D2 mutant) using His-tagged enzyme. The dissociation constants, K(a) and K(ia), of the mutant were similar to those of the wild-type enzyme but the catalytic constant, k(cat), was decreased to 28% that of the wild-type, indicating that some of the domain-domain interactions are lost due to the six-Lys insertion. Y68 plays a major role in arginine binding in the catalytic pocket in Limulus AK, and introduction of mutation at the Y68 position virtually abolishes catalytic activity. Thus, the constructed D1(Y68G)-D2 and D1-D2(Y68G) mutants mimic the D1(inactive)-D2(active) and D1(active)-D2(inactive) enzymes, respectively. The k(cat) values of both Y68 mutants were decreased to 13-18% that of the wild-type enzyme, which is much less than the 50% level of the two-domain enzyme. Thus, it is clear that substrate-binding to both domains is necessary for full expression of activity. In other words, substrate-binding appears to act as the trigger of the functional cooperativity in two-domain AK.
Tada
Hiroshi
H
Laboratory of Biochemistry, Faculty of Science, Kochi University, Kochi 780-8520 Japan.
Suzuki
Tomohiko
T
D000595:Amino Acid Sequence; D000818:Animals; D001122:Arginine Kinase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D055162:Biocatalysis; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012615:Sea Anemones / Q000201:enzymology*; D012995:Solubility
D021122:Protein Subunits; D001122:Arginine Kinase