Search

The active site of thermolysin-like proteases (TLPs) is located at the bottom of a cleft between the N- and C-terminal domains. Crystallographic studies have shown that the active-site cleft is more closed in Ligand-binding TLPs than in Ligand-free TLPs. Accordingly, it has been proposed that TLPs undergo a hinge-bending motion during catalysis resulting in "closure" and "opening" of the active-site cleft. Two hinge regions have been proposed. One is located around a conserved glycine 78; the second involves residues 135 and 136. The importance of conserved glycine residues in these hinge regions was studied experimentally by analyzing the effects of Gly --> Ala mutations on catalytic activity. Eight such mutations were made in the TLP of Bacillus stearothermophilus (TLP-ste) and their effects on activity toward casein and various peptide substrates were determined. Only the Gly78Ala, Gly136Ala, and Gly135Ala + Gly136Ala mutants decreased catalytic activity significantly. These mutants displayed a reduction in k(cat)/K-m for 3-(2-furylacryloyl)-L-glycyl-L-leucine amide of 73%, 62%, and 96%, respectively. Comparisons of effects on k(cat)/K-m for various substrates with effects on the K-i for phosphoramidon suggested that the mutation at position 78 primarily had an effect on substrate binding, whereas the mutations at positions 135 and 136 primarily influence k(cat). The apparent importance of conserved glycine residues in proposed hinge-bending regions for TLP activity supports the idea that hinge-bending is an essential part of catalysis.

Autolytic degradation of the thermolysin-like proteinase of Bacillus subtilis (TLP-sub) is responsible for the irreversible inactivation of the enzyme at elevated temperatures. Previously we have reported five cleavage sites in Tip-sub [Van den Burg et al, (1990) Biochem. J. 272, 93-97]. In an attempt to render the enzyme less susceptible to autolytic breakdown, one of the fission sites, located between Leu-156 and Ile-157, was modified by replacing Ile-157, C-terminally located with respect to the fission site, by an Asp residue. Aspartic acid is less preferred at this position with respect to the substrate preference of TLP-sub, Modelling studies indicated that this mutation was unlikely to cause conformational changes in the enzyme. Although the 156-157 fission was not observed in the mutant enzyme, a new fission site, between Gly-148 and Val-149, was now observed.

The present study concerns the use of site-directed muta- unfortunate in the case of NP-sub, since this enzyme is used genesis experiments to optimize a three-dimensional model in various industrial processes that could be improved if of the neutral protease of Bacillus subtilis (NP-sub). An rational manipulation of NP-sub properties would be possible. initial model of NP-sub was constructed using the crystal NP-sub-shares 47% sequence identity with thermolysin. structures of the homologous neutral proteases of Bacillus Studies of structures of homologous proteins (Chothia and thermoproteolyticus (thermolysin) and Bacillus cereus as Lesk, 1986; Sander and Schneider, 1991) suggest that a model templates. The largest portion of NP-sub could be modelled based on such a degree of sequence identity is often fairly satisfactorily, using standard techniques, but several accurate in the core region, but larger errors are to be expected surface-located regions could only be modelled with a in surface-located regions. A good model of the latter regions high degree of uncertainty. In order to make the model is of particular importance for engineering the stability of more reliable in these regions a ‘model building by muta- aspecific proteases such as subtilisin and NPs. Surface-located genesis’ approach was adopted. Mutations were designed regions are of major importance in the partial unfolding such that their effect on thermal stability could indicate processes that render the protease susceptible to autolysis and how their local environment should be modelled. This that are the rate-limiting step in the thermal inactivation approach provided insight in the local structure of several process (Dahlquist et al., 1976; Braxton and Wells, 1992; regions in NP-sub that were hard to model on the basis of Vriend and Eijsink, 1993; Kidokoro et al., 1995). This is homology with the two known structures alone. illustrated, for example, by the fact that the difference in

A 10 residue beta-hairpin, which is characteristic of thermostable Bacillus neutral proteases, was engineered into the thermolabile neutral protease of Bacillus subtilis. The recipient enzyme remained fully active after introduction of the loop. However, the mutant protein exhibited autocatalytic nicking and a 0.4 degree C decrease in thermostability. Two additional point mutations designed to improve the interactions between the enzyme surface and the introduced beta-hairpin resulted in reduced nicking and increased thermostability. After the introduction of both additional mutations in the loop-containing mutant, nicking was largely prevented and an increase in thermostability of 1.1 degrees C was achieved.