Cooperativity in the two-domain arginine kinase from the sea anemone Anthopleura japonicus

• Published in: International journal of biological macromolecules Vol. 42; no. 1; pp. 46 - 51
• Main Authors: Tada, Hiroshi, Nishimura, Yuko, Suzuki, Tomohiko
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 2008
• Subjects: Amino Acid Sequence; Amino Acid Substitution; Animals; Anthopleura (sea anemone); Arginine kinase; Arginine Kinase - biosynthesis; Arginine Kinase - chemistry; Arginine Kinase - genetics; Asparagine - chemistry; Asparagine - genetics; Catalysis; Cloning, Molecular; Cooperativity; Creatine kinase; Escherichia coli - genetics; Kinetic; Kinetics; Molecular Sequence Data; Protein Structure, Tertiary; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Sea Anemones - enzymology; Substrate Specificity; Synergism; Two-domain structure; Two-domain structure; Kinetic; Creatine kinase; Anthopleura (sea anemone); Arginine kinase; Cooperativity; Synergism
• ISSN: 0141-8130; 1879-0003
• DOI: 10.1016/j.ijbiomac.2007.09.004

cDNAs of the two-domain arginine kinase (AK) (contiguous dimer; denoted by 2D/WT) and its separated domains 1 and 2 (denoted by D1/WT and D2/WT) from the sea anemone Anthopleura japonicus, were cloned into the plasmid pMAL, and recombinant enzymes were expressed in E. coli as MBP fusion proteins. The kinetic parameters k cat, K a and K ia, were determined for all three AKs. All three enzymes showed distinct AK activity, and had high affinity for arginine ( K a Arg = 0.25 – 0.48   mM ). The catalytic efficiency, calculated by k cat / K a Arg K ia ATP , of the 2D/WT enzyme (182 mM −2 s −1, the value for one active 40 kDa domain) was two- to three-times higher than values for either D1/WT or D2/WT (80.2 and 86.4 mM −2 s −1, respectively), suggesting the presence of domain-domain interactions (cooperativity) in the contiguous dimer. The K ia/ K a values of the three enzymes ranged from 0.88 to 1.32, indicating that there is no strong synergism in substrate binding, as seen in typical AKs. Asp62 and Arg193, which are conserved in most AKs and play a key role in stabilizing the substrate-bound structure, are also conserved in the two domains of Anthopleura AK. We replaced Asp62 in D2/WT with Glu or Gly. The catalytic efficiency and K ia/ K a for the D62E mutant were comparable to those of D2/WT, but catalytic efficiency for the D62G mutant was decreased to 13% of that of the D2/WT with a significantly increased value of K ia/ K a (1.92), indicating that Asp62 plays an important role in the expression of AK activity.