Cooperativity in the two-domain arginine kinase from the sea anemone Anthopleura japonicus.
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 kcat, Ka and Kia, were determined for all three AKs. All three enzymes showed distinct AK activity, and had high affinity for arginine (Ka Arg=0.25-0.48 mM). The catalytic efficiency, calculated by kcat/Ka ArgKia 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 86.4mM(-2)s(-1), respectively), suggesting the presence of domain-domain interactions (cooperativity) in the contiguous dimer. The Kia/Ka 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 Kia/Ka 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 Kia/Ka (1.92), indicating that Asp62 plays an important role in the expression of AK activity.
Tada
Hiroshi
H
Laboratory of Biochemistry, Faculty of Science, Kochi University, Kochi 780-8520 Japan.
Nishimura
Yuko
Y
Suzuki
Tomohiko
T
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001122:Arginine Kinase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D001216:Asparagine / Q000737:chemistry / Q000235:genetics; D002384:Catalysis; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012615:Sea Anemones / Q000201:enzymology*; D013379:Substrate Specificity
D011993:Recombinant Fusion Proteins; D001216:Asparagine; D001122:Arginine Kinase