The roles of C-terminal loop residues of dimeric arginine kinase from sea cucumber Stichopus japonicus in catalysis, specificity and structure

Abstract

Arginine kinase (AK) catalyzes the reversible phosphorylation of arginine by MgATP to form a high-energy compound phosphoarginine (Parg) and MgADP in forward reaction in invertebrates. To detect the different catalytical mechanisms among Stichopus-AK (dimer) and Limulus-AK (monomer) and Torpedo creatine kinase (dimeric CK) and to reveal the structural role of the C-terminal domain loop (C-loop) of dimeric AK, six single-site mutants, E314D, E314Q, E314V, F315A, F315H and F315Y were constructed as well as two multi-site variants, S312R/F315H/V319E (formed by substituting the C-loop of monomeric AK for that of dimeric AK, termed the AAloop) and S312G/E314V/F315D/E317A/S318A/G321S (formed by substituting the C-loop of dimeric CK for that of dimeric AK, termed the ACloop). The AK activity of the three mutants at Glu 314 decreased significantly, from 60- to 500-fold. The ACloop showed only slight AK activity, unlike the same construction in Limulus-AK. In addition, all Phe 315 mutants including the AAloop which retained Glu 314 had modest AK activity (5–84% of the wild type). All the results above suggested that Glu 314 played a more significant role in catalysis in dimeric AK than in the monomer. In addition, ANS profiles indicated that the tolerance of the three Glu 314 mutants to denaturant decreased slightly compared with wild type AK. Though monomeric AK has a His residue at site 315, mutants F315H and the AAloop could not resist any perturbation of denaturant, and the mutants showed a Gibbs free energy of about 2.7 kJ/mol lower than wild type AK. Therefore Phe 315 in dimeric AK has a different role from His 315 in monomeric AK. This might contribute to the stabilization of the native conformation, while His 315 in Limulus AK directly binded to the carboxylate of arginine. Taking all the results above together, we suggested a unique mechanism in dimeric AK, different from both monomeric AK and dimeric CK.