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Abstract: Arginine kinase (AK), a crucial enzyme for the energy metabolism of invertebrates, catalyzes the reversible phosphorylation of arginine by Mg2+ATP to form phosphoarginine and Mg2+ADP. Arginine 330 (R330), not involved in the catalysis of phosphoryl transfer, is a residue highly conserved in the phosphagen kinase family. In order to investigate the role of R330 in AK, it was replaced by lysine (R330K). Non-reduced SDS-PAGE analysis suggested that wild type AK (Wt-AK) and R330K existed in two forms, the reduced form (R-AK or R-R330K) and the oxidized form (O-AK or O-R330K), whereas O-R330K was more susceptible to generate than O-AK. Subsequently, an intramolecular disulfide bond in O-R330K was demonstrated to be formed between Cys201 and Cys271 by site-directed mutagenesis. Biochemical analysis revealed that conformational changes of R330K were concomitant with the sharp decline of catalytic activity. These results were further confirmed by structure modeling of AK and R330K. Therefore, it can be concluded that R330 residue plays an important role in the structural stability and activity of AK. [Copyright &y& Elsevier]

Abstract: The purpose of this study is to clarify that the amino acid residues (Asp62 and Arg193) are responsible for the activity and stability of arginine kinase (AK). The amino acid residues Asp62 (D62) and Arg193 (R193) are strictly conserved in monomeric AKs and form an ion pair in the transition state analogue complex. In this research, we replaced D62 with glutamate (E) or glycine (G) and R193 with lysine (K) or glycine (G). The mutants of D62E and R193K retained almost 90% of the wild-type activity, whereas D62G and R193G had a pronounced loss in activity. A detailed comparison was made between the physic-chemical properties and conformational changes of wild-type AK and the mutants by means of ultraviolet (UV) difference and fluorescence spectra. The results indicated that the conformation of all of the mutants had been changed and the stability in a urea solution was also reduced. We speculated that the hydrogen bond and electrostatic interactions formed between residues 62 and 193 play a key role in stabilizing the structure and mediating the synergism in substrate binding of arginine kinase from greasyback shrimp (Metapenaeus ensis). [Copyright &y& Elsevier]

Abstract: Arginine kinase (AK), a crucial enzyme in energy metabolism, buffers cellular ATP levels by catalyzing the reversible phosphoryl transfer between ATP and arginine. To better understand the role of Cys271 in conformational changes of AK from greasyback shrimp (Metapenaeus ensis), we replaced the residue with serine and alanine. A detailed comparison of the catalytic activity and conformation was made between wild-type AK and the mutants by means of activity analysis, ultraviolet (UV) difference, fluorescence spectrum and size exclusion chromatography (SEC). The results indicated that the catalytic activity of the two mutants was gone. The substrates, arginine–ADP–Mg2+ could induce conformational changes, and additional NO3 − could induce further changes in both the native enzyme and the variants. We speculated that Cys271 might be located in the hinge region between the two domains of AK and cause enzyme conformational changes upon addition of substrate. [Copyright &y& Elsevier]