Evidence that the amino acid residue Ile121 is involved in arginine kinase activity and structural stability.

Arginine kinase (AK) catalyzes the reversible phosphorylation of arginine by ATP, yielding the phosphoarginine. Domain-domain interactions may be very important to the structure and functions of many multidomain proteins. However, little is known about the role of amino acid residues located in the linker between the N- and C-terminal domains in the structural stability and functions of multidomain proteins. In this research, A series mutation of conserved residue Ile121 located in the linker were mutated to explore its roles in the activity and structural stability of AK. The mutations I121D and I121K led to pronounced loss of activity and structural stability. Furthermore, these mutations also led to serious aggregation during heat-and GdnHCl-induced denaturation and refolding from the GdnHCl-denatured state. More importantly, all the mutantions except I121L could not successfully recover their activities by dilution-initiated refolding, and showed significant decreased rate constant during AK refolding. While the mutation I121L almost had no effect on AK activity and structural stability. These results suggested that mutations of the residue I121 in the linker might affect the correct positioning of the domains and thus disrupt the efficient recognition and interactions between the N- and C-terminal domains.