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Arginine kinase (AK) catalyzes the reversible phosphorylation of arginine by ATP, yielding the phosphoarginine. Amino acid residues in the guanidine specificity (GS) region play important roles in the guanidine-recognition. However, little is known about roles of amino acid residue G66 in the GS region in proteins folding, activity and structural stability. In this study, a series of G66 mutations were constructed to investigate its roles in AK's structural stability and activity. Our studies revealed that mutations in this conserved site could cause pronounced loss of activity, conformational changes and structural stability. Spectroscopic experiments indicate that G66 mutations influences AK transition from the molten globule intermediate to the native state in folding process. These results provided herein may suggest that amino acid residue G66 may play a relatively important role in AK's activity and structural stability. [ABSTRACT FROM AUTHOR]

Abstract: Arginine kinase (AK) is a key enzyme in cellular energy metabolism of invertebrates. There are two conserved amino acid residues D14 and R138 in dimeric AK which form inter-subunit hydrogen bond. In Stichopus japonicus AK, mutations in these residues caused pronounced loss of activity, conformational changes and distinct substrate synergism alteration. Mutations (R138G, R138A and D14G) abolished D14 and R138 interaction disrupted the structure or conformation of S. japonicus AK. These R138G, R138A and D14G mutations changed their native assembles of dimeric AK and caused them in a partially unfolded state. The partially unfolded state of these mutant AKs made them prone to aggregate under environmental stress. The D14E/R138K and R138K mutant AKs showed similar characteristics to those of WT AK for forming the interaction which could replacement roles of D14 and R138 interaction. These results suggested that D14 and R138 interaction is involved in AK's activity, substrate synergism and structural stability. [Copyright &y& Elsevier]

Abstract: Arginine kinase (AK) is a key enzyme for cellular energy metabolism, catalyzing the reversible phosphoryl transfer from phosphoarginine to ADP in invertebrates. The amino acid residue C271 is involved in keeping AK's activity and constraining the orientation of the substrate arginine. However, the roles of the C271 interaction amino acid residues in AK's substrate synergism, activity and structural stability are still unclear. The crystal structure of AK implied that the amino acid residue T273 interacted with the residue C271 and might play vital roles in keeping AK's activity, substrate synergism and structural stability. The mutations T273G and T273A led to significantly loss of activity, obviously decreased of substrate synergism and structural stability. Furthermore, spectroscopic experiments indicated that mutations T273G and T273A impaired the structure of AK and led them to a partially unfolded state. The inability to fold to the functional state made the mutations prone to aggregate under environmental stresses. Moreover, the mutations T273S and T273D almost had no effects on AK's activity and structural stability. This study herein indicated that the residue T273 played key roles in AK's activity, substrate synergism and structural stability. [Copyright &y& Elsevier]