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.
Nyman
Tomas
T
Department of Cell Biology The Wenner-Gren Institute, Stockholm University, Stockholm, S-106 91, Sweden.
Schüler
Herwig
H
Korenbaum
Elena
E
Schutt
Clarence E
CE
Karlsson
Roger
R
Lindberg
Uno
U
D000199:Actins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001704:Biopolymers / Q000737:chemistry / Q000378:metabolism; D002645:Chickens / Q000235:genetics; D003850:Deoxyribonuclease I / Q000378:metabolism; D004795:Enzyme Stability; D006639:Histidine / Q000378:metabolism*; D006860:Hydrogen Bonding; D006868:Hydrolysis; D007700:Kinetics; D008745:Methylation; D015879:Myosin Subfragments / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013696:Temperature; D013816:Thermodynamics; D014783:Viscosity
D000199:Actins; D001704:Biopolymers; D015879:Myosin Subfragments; D010710:Phosphates; D006639:Histidine; D000255:Adenosine Triphosphate; D003850:Deoxyribonuclease I; D000251:Adenosine Triphosphatases