Two-domain arginine kinase from the deep-sea clam Calyptogena kaikoi—Evidence of two active domains

Abstract

The cDNA and deduced amino acid sequences for arginine kinase (AK) from the deep-sea clam Calyptogena kaikoi have been determined revealing an unusual two-domain (2D) structure with molecular mass of 80 kDa, twice that of normal AK. The amino acid sequences of both domains contain most of the residues thought to be required for substrate binding found in the horseshoe crab Limulus polyphemus AK, a well studied system for which several X-ray crystal structures exist. However, two highly conserved residues, D62 and R193, that form a salt bridge thereby stabilizing the substrate-bound structure have been replaced by G and N in domain 1, and G and P in domain 2, respectively. The present effort probes whether both domains of Calyptogena AK are catalytically competent. Recombinant constructs of the wild-type enzyme of both single domains, and of selected mutants of the Calyptogena AK have been expressed as fusion proteins with the maltose-binding protein. The wild-type two-domain enzyme (2D[WT]) had high AK activity ( k cat = 23 s − 1, average value of the two domains), and the single domain 2 (D2[WT]) showed 1.5-times higher activity ( k cat = 38 s − 1) than the wild-type 2D[WT]. Interestingly, the single domain 1 (D1[WT]) showed only a very low activity ( k cat ∼ 0.016 s − 1). Introduction of a Y68A mutation in both domains virtually abolished catalytic activity. On the other hand, significant residual activity was observed ( k cat = 2.8 s − 1), when the Y68A mutation was introduced only into domain 2 of the two-domain enzyme. A similar mutation in domain 1 of the two-domain enzyme reduced activity to a much lower extent ( k cat = 11.1 s − 1). Although the domains of this “contiguous” dimeric AK each have catalytic capabilities, the presence of domain 2 strongly influences the stability and activity of domain 1.