Mutational analysis of arginine 177 in the nucleotide binding site of beta-actin

Actin ADP-ribosylated at arginine 177 is unable to hydrolyze ATP, and the R 177 side chain is in a position similar to that of the catalytically essent ial lysine 71 in heat shock cognate protein Hsc70, another member of the ac tin-fold family of proteins. Therefore, actin residue R177 has been implica ted in the mechanism of ATP hydrolysis. This paper compares wild-type beta- actin with a mutant in which R177 has been replaced by aspartic acid. The m utant beta-actin was expressed in Saccharomyces cerevisiae and purified by DNase I-affinity chromatography. The mutant protein exhibited a reduced the rmal stability and an increased nucleotide exchange rate, suggesting a weak ened interdomain connection. The ATPase activity of G-actin and the ATPase activity expressed during polymerization were unaffected by the R177D repla cement, showing that this residue is not involved in catalysis. In the pres ence of polymerizing salts, ATP hydrolysis by both wild-type Mg-beta-actin and the mutant protein preceded filament formation. With the mutant actin, the initial rate of ATP hydrolysis was as high as with wild-type actin, but polymer formation was slower, reached lower steady-state levels, and the p olymers formed exhibited much lower viscosity. The critical concentration o f polymerization (A(cc)) of the mutant actin was increased 10-fold as compa red to wild-type actin. Filaments formed from the R177D mutant beta-actin b ound phalloidin.