The role of MeH73 in actin polymerization and ATP hydrolysis.

In actin from many species H73 is methylated, but the function of this rare post-translational modification is unknown. Although not within bonding distance, it is located close to the gamma-phosphate of the actin-bound ATP. In most crystal structures of actin, the delta1-nitrogen of the methylated H73 forms a hydrogen bond with the carbonyl of G158. This hydrogen bond spans the gap separating subdomains 2 and 4, thereby contributing to the forces that close the interdomain cleft around the ATP polyphosphate tail. A second hydrogen bond stabilizing interdomain closure exists between R183 and Y69. In the closed-to-open transition in beta-actin, both of these hydrogen bonds are broken as the phosphate tail is exposed to solvent. Here we describe the isolation and characterization of a mutant beta-actin (H73A) expressed in the yeast Saccharomyces cerevisiae. The properties of the mutant are compared to those of wild-type beta-actin, also expressed in yeast. Yeast does not have the methyl transferase necessary to methylate recombinant beta-actin. Thus, the polymerization properties of yeast-expressed wild-type beta-actin can be compared with normally methylated beta-actin isolated from calf thymus. Since earlier studies of the actin ATPase almost invariably employed rabbit skeletal alpha-actin, this isoform was included in these comparative studies on the polymerization, ATP hydrolysis, and phosphate release of actin. It was found that H73A-actin exchanged ATP at an increased rate, and was less stable than yeast-expressed wild-type actin, indicating that the mutation affects the spatial relationship between the two domains of actin which embrace the nucleotide. At physiological concentrations of Mg(2+), the kinetics of ATP hydrolysis of the mutant actin were unaffected, but polymer formation was delayed. The comparison of methylated and unmethylated beta-actin revealed that in the absence of a methyl group on H73, ATP hydrolysis and phosphate release occurred prior to, and seemingly independently of, filament formation. The comparison of beta and alpha-actin revealed differences in the timing and relative rates of ATP hydrolysis and P(i)-release.