Identification of the key amino acid residues in Sabellastarte arginine kinase for distinguishing chiral guanidino substrates (d- and l-arginine)

Arginine kinase (AK, EC 2.7.3.3) is a phosphagen kinase widely distributed in lower and higher invertebrates. Sabellastarte indica AKs (AK1 and AK2) are the only AKs that use d-arginine in addition to l-arginine as a substrate. S. indica AKs are therefore clearly unrelated to typical AK. The purpose of this work was to identify the amino acid residues responsible for distinguishing the chirality of d- and l-arginine. The three-dimensional structure of Sabellastarte AK2 was predicted using the SWISS-MODEL automated modeling server. Two amino acids, L64 in the N-terminal flexible loop and N320 in the C-terminal flexible loop, were found to be closest to the substrate arginine. We introduced several mutations around the predicted binding site of the substrate arginine. In the L64I mutant, the affinity for l-arginine was greatly increased (9.5-fold that of the wild-type), whereas its affinity for d-arginine was increased 2.9-fold, indicating that the L64I mutant enzyme was more specific for l-arginine. On the other hand, the L64V mutant showed a 1.7-fold decrease in affinity for l-arginine, but unchanged affinity for d-arginine. Thus the L64V mutant enzyme was more specific for d-arginine. Since the replacement of amino acid residue L64 by I or V significantly affected the affinity for l-arginine or d-arginine, it can be concluded that amino acid 64 is a key residue for distinguishing d- and l-arginine. Seven mutants at position 320 (N320H, Q, D, E, R, K and A) had considerably reduced enzymatic activity (0.05–52% of the wild-type). The reduction in enzyme activity was more significant when d-arginine was the substrate rather than l-arginine, except for N320D, suggesting that the carbonyl oxygen of the Asn320 side chain forms a hydrogen bond with the α-amino nitrogen attached to the asymmetric carbon of the d-arginine substrate. In addition, we found that even a residue remote from the guanidino substrate-binding site, such as G54 and Y89 in Sabellastarte AK2, significantly affected guanidino substrate specificity.