COMPAAN, D.M.*; ELLINGTON, W.R.: Cloning, Expression and Characterization of a Contiguous, Dimeric Arginine Kinase

Arginine kinase (AK) plays a central role in cellular energy homeostasis in a variety of invertebrates. The foot muscle of the razor clam Ensis directus contains extraordinarily high activities of AK [~1,000 enzyme units per g]. We have isolated this AK; the subunit and native relative molecular masses (Mr) are identical, on the order of 82 kD which is roughly twice the native Mr values of typical AKs. We have cloned the cDNA for Ensis AK and expressed the recombinant protein in E. coli. This AK consists of two enzyme domains fused together to form a “contiguous dimer”. Sequence comparisons suggest that this duplication/fusion event occurred after the divergence of the bivalves from other members of the molluscan lineage. Sequence analyses reveal the presence of two catalytic sites which differ somewhat in terms of key residues placing into question whether there is equivalence in terms of activity. Detailed kinetic analyses (binary [Ks] and ternary [Km] binding constants, true Vmax values) of the recombinant Ensis AK showed that this enzyme has a true Vmax in the same range as that of recombinant AK from the horseshoe crab (which is monomeric). Ks and corresponding Km values were virtually identical (Ks/Km = ~1) for Ensis AK indicating no (or even slightly negative) synergism of substrate binding. In contrast, the horseshoe crab AK displayed pronounced synergism of substrate binding (Ks/Km > 3). While Ensis AK retains high activity, we hypothesize that the unique fusion of two enzyme domains places constraints on the structure of the protein thereby restricting conformational movements associated with substrate binding. (Supported by NSF grant IBN-9631907 to WRE).