Kinetic analysis of two purified forms of arginine kinase: absence of cooperativity in substrate binding of dimeric phosphagen kinase.

Arginine kinase from sea urchin eggs and sea cucumber muscle are dimeric enzymes, unlike the more widely distributed monomeric enzyme found in other invertebrates. Both purified enzymes exhibited features characteristic of the monomeric arginine kinases including pH optima, formation of a catalytic dead-end complex (enzyme-MgADP-arginine) and stabilization of this complex by monovalent anions. A complete analysis of initial velocity data, in both directions for each substrate, indicated that substrate binding cooperativity was either minimal or non-existent. Unlike many other multi-subunit enzymes, the significance of the dimeric state of the phosphagen kinases remains unclear. These present results would suggest that (a) cooperativity, or so-called synergism in substrate binding is not a characteristic of the dimeric state of the protein and (b) the functional significance of the dimeric state is not related to the ability of some of these enzymes to undergo cooperativity in substrate binding. The significance of the dimeric state for the creatine kinases and arginine kinases remains to be established.

Immunological and physical comparison of monomeric and dimeric phosphagen kinases: Some evolutionary implications.

The antigenic and physical properties of several representative invertebrate phosphagen kinases have been examined in order to further characterize the relationship between taxonomic assignment, quaternary protein structure and evolution of this class of enzymes. Antibodies against dimeric arginine kinase from the sea cucumber cross-reacted with dimeric arginine kinase purified from sea urchin eggs, but failed to react with extracts from any species known to contain monomeric arginine kinase. However, strong immunoreactivity was observed when antibodies against purified dimeric arginine kinase were reacted with pure creatine kinase from the human muscle (CK-MM) and brain (CK-BB) as well as extracts from several species known to contain dimeric creatine kinase. Of particular interest with regard to evolution of the phosphagen kinases, we confirm the presence of creatine kinase activity in the very primitive sponge Tethya aurnatium and detect a reaction with antibodies against dimeric, but not monomeric, arginine kinase. This observation is consistent with recent studies of phosphagen kinase evolution. Substrate utilization was very specific with creatine kinase using only creatine. Arginine kinase catalyzed phosphorylation of arginine but enzymes from several species could also phosphorylate canavanine. No activities were detected with d-arginine. Isoelectric points, evaluated for several pure arginine kinases suggest that generally the monomeric forms are more acidic than the dimeric proteins. Heat inactivation of arginine kinase in several species indicated a wide range of stabilities, which did not appear to be correlated with quaternary structure, but rather distinguished by the organism's environment. On the other hand, homodimeric arginine kinase proteins from species inhabiting disparate environments are sufficiently homologous to form a catalytically active hybrid.