The members of the M4 peptidase family are involved in processes as diverse as pathogenicity and industrial applications. For the first time a number of M4 family members, also known as thermolysin-like proteases, has been characterized with an identical substrate set and a uniform set of assay conditions. Characterization with peptide substrates as well as high performance liquid chromatography analysis of ␤-casein digests shows that the M4 family is a homogeneous family in terms of catalysis, even though there is a significant degree of amino acid sequence variation. The results of this study show that differences in substrate specificity within the M4 family do not correlate with overall sequence differences but depend on a small number of identifiable amino acids. Indeed, molecular modeling followed by site-directed mutagenesis of one of the substrate binding pocket residues of the thermolysin-like proteases of Bacillus stearothermophilus converted the catalytic characteristics of this variant into that of thermolysin.

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The impact of long range electrostatic interactions on catalysis in the thermolysin-like protease from Bacillus stearothermophilus was studied by analyzing the effects of inserting or removing charges on the protein surface. Various mutations were introduced at six different positions, and double-mutant cycle analysis was used to study the extent to which mutational effects were interdependent. The effects of single point mutations on the k cat /K m were non-additive, even in cases where the point mutations were located 10 Å or more from the active site Zn 2؉ and separated from each other by up to 25 Å. This shows that catalysis is affected by large electrostatic networks that involve major parts of the enzyme. The interdependence of mutations at positions as much as 25 Å apart in space also indicates that other effects, such as active site dynamics, play an important role in determining active site electrostatics. Several mutations yielded a significant increase in the activity, the most active (quadruple) mutant being almost four times as active as the wild type. In some cases the shape of the pH-activity profile was changed significantly. Remarkably, large changes in the pH-optimum were not observed.The acceleration of reaction rates by enzymes is one of the essential prerequisites for life as we know it, and the multitude and diversity of enzymes shows that it should be possible to design an enzyme that will catalyze almost any reaction under almost any set of conditions. To achieve a high rate of acceleration, enzymes rely on charged groups in their active site that stabilize the transition state or function as acid or base catalysts in the reaction. The kinetic parameters of enzymes therefore display a significant pH dependence, which is determined by the pK a values of the active site groups.Catalysis depends on intricate electrostatic interactions, which may be noticeable over distances that are large compared with short range of interactions such as hydrogen bonds and hydrophobic contacts. Thus, larger parts of an enzyme may be involved in optimizing its catalytic center than previously thought. The long range character of electrostatic effects is illustrated by a, very limited, number of examples in the literature, showing that changes in surface charge at locations as far as 15 Å from a catalytic center may affect enzyme activity (1, 2). Unfortunately, electrostatic interactions are hard to handle theoretically not only because of their long range character but also because of intrinsic theoretical difficulties. For example, most electrostatic models still use a single rigid protein structure and, at most, two dielectric constants to account for all the dynamics of the protein. This clearly is an oversimplification of reality (3).We have studied the contribution of long range electrostatic interactions to catalysis by analyzing the effects of a series of charge mutations scattered over a larger part of the surface of a thermolysin-like protease from Bacillus stearothermophilus (TLP-ste).

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 kcat/Km for 3-(2-furylacryloyl)-L-glycyl-L-leucine amide of 73%, 62%, and 96%, respectively. Comparisons of effects on kcat/Km for various substrates with effects on the Ki 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 kcat. 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.