PubMed2002
By mimicking the partial spatial structure of the dimer of the l-aspartase subunit, the central ten-helix bundle, and an "active site" between the cleft of domain 1 (D1) and domain 3 (D3) from different subunits, we designed l-aspartase variants, in which D1D2 and D2D3 were ligated with a random hexapeptide loop. As expected, we obtained the variant with the highest activity (relative activity is 21.3% of the native enzyme, named as drAsp017) by in vitro selection. The molecular weight of this variant, obtained from size-exclusion column chromatography, is about 81 kDa, which indicates that it is indeed a monomer, whereas native l-aspartase is a tetramer. The activity-reversibility of drAsp017 (10(-7) m) was 80% after incubation for 30 min at 50 degrees C, while native enzyme only retained about 17% under the same conditions. Reactivation of drAsp017 denatured in 4 m guanidine HCl was independent of protein concentration at up to 20 x 10(-8) m at 25 degrees C, whereas the protein concentration of native enzyme strongly affected its reactivation under the above conditions. The sensitivity of drAsp017 (10(-7) m) to effective factors in the fumarate-amination reaction compared with native enzyme was also determined. Half-saturating concentrations of the activator l-aspartate and Mg2+ for drAsp017 (0.8 and 0.5 mm, respectively) are much higher than that of the native enzyme (0.10 and 0.15 mm, respectively). The data show that a monomeric l-aspartase is obtained by in vitro selection. Thus, the conversion of oligomeric proteins into their functional monomers could have important applications.