FIGURE 7.

Arg⁴⁴⁰ and Glu⁵³⁷ can be in close proximity in the KC and these residues are essential for autophosphorylation. A, mutation of Arg⁴⁴⁰ and Glu⁵³⁷ to cysteines induced disulfide bond formation between the two residues. Although wild-type KC and four mutants (C524S, C524S/R440C, C524S/E537C, and C524S/R440C/E537C) were shown as a single band by SDS-PAGE in the presence of dithiothreitol, dimer-sized bands were detected in WT, C524S/E537C, and C524S/R440C/E537C in the absence of the reducing agent. The presence of multiple bands in the C524S/R440C/E537C mutant indicates various interactions between Cys⁴⁴⁰ and Cys⁵³⁷ in a KC dimer. B, mutation of Arg⁴⁴⁰ or Glu⁵³⁷ leads to a loss of the autophosphorylation activity compared the C524S mutant; activity of the R440C mutant was completely ablated.