Efficient transesterification of sucrose catalysed by the metalloprotease thermolysin in dimethylsulfoxide.

Thermolysin catalyses the formation of sucrose esters from sucrose and vinyl laurate in dimethylsulfoxide, with a specific activity of 53 nmol/min/mg and 2-O-lauroyl-sucrose as the main product. Such transesterification reactions are normally observed only when the mechanism involves an acyl enzyme intermediate, as with lipases or serine proteases, and not with metalloproteases like thermolysin. A possible reason is the affinity of the active site of thermolysin for sugar moieties, as for the potent inhibitor phosphoramidon.

The effect of changing the hydrophobic S1' subsite of thermolysin-like proteases on substrate specificity.

The hydrophobic S1' subsite is one of the major determinants of the substrate specificity of thermolysin and related M4 family proteases. In the thermolysin-like protease (TLP) produced by Bacillus stearothermophilus (TLP-ste), the hydrophobic S1' subsite is mainly formed by Phe130, Phe133, Val139 and Leu202. In the present study, we have examined the effects of replacing Leu202 by smaller (Gly, Ala, Val) and larger (Phe, Tyr) hydrophobic residues.

A single calcium binding site is crucial for the calcium-dependent thermal stability of thermolysin-like proteases.

Thermostable thermolysin-like proteases (TLPs), such as the TLP of Bacillus stearothermophilus CU-21 (TLP-ste), bind calcium in one double (Ca1,2) and two single (Ca3, Ca4) calcium binding sites. The single sites are absent in thermolabile TLPs, suggesting that they are determinants of (variation in) TLP stability. Mutations in the Ca3 and Ca4 sites of TLP-ste indeed reduced thermal stability, but only mutations in the Ca3 site affected the calcium-dependence of stability.

Probing catalytic hinge bending motions in thermolysin-like proteases by glycine --> alanine mutations.

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.

Rendering one autolysis site in Bacillus subtilis neutral protease resistant to cleavage reveals a new fission.

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 TLP-sub [Van den Burg et al. (1990) Biochem.