Alpha-actinin structure and regulation.

Alpha-actinin is a cytoskeletal actin-binding protein and a member of the spectrin superfamily, which comprises spectrin, dystrophin and their homologues and isoforms. It forms an anti-parallel rod-shaped dimer with one actin-binding domain at each end of the rod and bundles actin filaments in multiple cell-type and cytoskeleton frameworks. In non-muscle cells, alpha-actinin is found along the actin filaments and in adhesion sites. In striated, cardiac and smooth muscle cells, it is localized at the Z-disk and analogous dense bodies, where it forms a lattice-like structure and stabilizes the muscle contractile apparatus. Besides binding to actin filaments alpha-actinin associates with a number of cytoskeletal and signaling molecules, cytoplasmic domains of transmembrane receptors and ion channels, rendering it important structural and regulatory roles in cytoskeleton organization and muscle contraction. This review reports on the current knowledge on structure and regulation of alpha-actinin.

Enhancement of catalytic efficiency by the combination of site-specific mutations in a carbonic anhydrase-related protein.

A single mutation, involving the replacement of an arginine residue with histidine to reconstruct a zinc-binding site, suffices to change a catalytically inactive murine carbonic anhydrase-related protein (CARP) to an active carbonic anhydrase with a CO2-hydration turnover number of 1.2 x 104 s-1. Further mutations, leading to a more 'carbonic anhydrase-like' active-site cavity, results in increased activity. A quintuple mutant having His94, Gln92, Val121, Val143, and Thr200 (human carbonic anhydrase I numbering system) shows kcat = 4 x 104 s-1 and kcat/Km = 2 x 107 M-1.s-1, greatly exceeding the corresponding values for carbonic anhydrase isozyme III and approaching those characterizing carbonic anhydrase I. In addition, a buffer change from 50 mM Taps/NaOH to 50 mM 1, 2-dimethylimidazole/H2SO4 at pH 9 results in a 14-fold increase in kcat for this quintuple mutant. The CO2-hydrating activity of a double mutant with His94 and Gln92 shows complex pH-dependence, but the other mutants investigated behave as if the activity (kcat/Km) is controlled by the basic form of a single group with pKa near 7.7. In a similar way to human carbonic anhydrase II, the buffer behaves formally as a second substrate in a ping-pong pattern, suggesting that proton transfer between a zinc-bound water molecule and buffer limits the maximal rate of catalysis in both systems at low buffer concentrations. However, the results of isotope-exchange kinetic studies suggest that proton shuttling via His64 is insignificant in the CARP mutant in contrast with carbonic anhydrase II. The replacement of Ile residues with Val in positions 121 or 143 results in measurable 4-nitrophenyl acetate hydrolase activity. The pH-rate profile for this activity has a similar shape to those of carbonic anhydrase I and II. CD spectra of the double mutant with His94 and Gln92 are variable, indicating an equilibrium between a compact form of the protein and a 'molten globule'-like form. The introduction of Thr200 seems to stabilize the protein.

Changing the efficiency and specificity of the esterase activity of human carbonic anhydrase II by site-specific mutagenesis.

Rates of hydrolysis of 4-, 3-, and 2-nitrophenyl acetate and 4-nitrophenyl propionate catalyzed by wild-type and mutant forms of human carbonic anhydrase II have been measured. The results show that the mutations Tyr7-->Phe and Ala65-->Leu lead to activity enhancements with all the investigated substrates, but there is no significant effect on the specificity. In contrast, some mutations at sequence position 200 have large effects on specificity. For example, while the mutation Thr200-->Gly results in a threefold increase of the rate of hydrolysis of 4-nitrophenyl acetate, the activity is enhanced 10 times with the meta-substituted substrate and 380 times with the ortho-substituted substrate. These results are interpreted in terms of the removal in the mutant of a steric interference between the 2-NO2 group, in particular, and the side chain of Thr200. Mutants involving residues lining a hydrophobic pocket near the catalytically essential zinc ion have also been investigated. The most pronounced effect on specificity was found for the Val143-->Gly mutant. This mutation leads to a sixfold decrease of the rate of hydrolysis of 4-nitrophenyl acetate but a 20-fold increase of the activity with the propionyl ester as substrate. These results suggest that the side chain of Val143 interferes sterically with the acyl moiety of 4-nitrophenyl propionate. Based on these results, we have constructed a hypothetical model of the location of these ester substrates in the enzymic active site.

Two point mutations convert a catalytically inactive carbonic anhydrase-related protein (CARP) to an active enzyme.

A murine carbonic anhydrase-related protein (CARP) has been expressed in Escherichia coli and purified to near homogeneity. The polypeptide chain consists of 290 amino acid residues and has a calculated molecular mass of 32,950 Da. By introducing two mutations, Arg117 --> His and Glu115 --> Gln, we created a metal-binding center homologous to that in the carbonic anhydrases from the animal kingdom. In contrast to unmodified CARP, this double mutant was isolated as a 1:1 zinc-protein complex. While unmodified CARP is catalytically inactive, the mutant catalyzes CO2 hydration with a significantly higher efficiency than the mammalian low-activity carbonic anhydrase isozyme III. The activity is strongly inhibited by the powerful and selective carbonic anhydrase inhibitor, acetazolamide.