On the basis of the homology with the Bacillus thermoproteolyticus zinc endopeptidase thermolysin, we hypothesized that Glu-143 and His-231 are the key residues for the catalytic activity of the Bacillus subtilis neutral protease. To test this possibility by site-directed mutagenesis, we substituted these two residues with Ala, Ser, Trp and Arg, and Leu, Val and Cys respectively. All these substitutions dramatically affected the amount of secreted mutant proteins, as determined by immunological methods, and their catalytic activities. No appreciable secretion was observed with the three Glu mutants Trp, Ser and Arg, whereas the Glu----Ala mutant enzyme was secreted at a level of a few hundred micrograms per litre of culture. The His mutants were all secreted at higher levels (in the order of a few milligrams per litre) and their residual catalytic activity could be determined using Z-Ala-Leu-Ala as substrate. Our results confirm the key role played by Glu-143 and His-231 in catalysis and moreover suggest the existence of a relationship between the catalytic activity of the enzyme and the extent of its secretion. In this context, we present data suggesting an autoproteolytic mechanism of cleavage of the precursor form of the enzyme, analogous to the one previously reported for the B. subtilis subtilisin.

The surface loop which in the Bacillus subtilis neutral protease (NP) extends from amino acid residue 188 to residue 194 was replaced, by site-directed mutagenesis, with the 10-residue segment which in the homologous polypeptide chain of thermolysin (TLN) binds calcium-4 [Matthews, B. W., Weaver, L. H., & Kester, W. R. (1974) J. Biol. Chem. 249, 8030-8044]. The mutant NP was isolated to homogeneity, and its structural, functional, calcium-binding, and stability properties were investigated. Proteolytic fragmentation with Staphylococcus aureus V8 protease of mutant NP was used to isolate and analyze the protein fragment encompassing the site of mutation, unambiguously establishing the effective insertion of the new 10-residue segment. Atomic absorption measurements allowed us to demonstrate that mutant NP binds three calcium ions instead of the two ions bound to wild-type NP, showing that indeed the chain segment grafted from TLN to NP maintains its calcium-binding properties. The mutant NP showed kinetic parameters essentially similar to those of the wild-type NP with Z-Phe-Leu-Ala-OH as substrate. The enzyme inactivation of mutant vs wild-type NP was studied as a function of free [Ca2+]. It was found that mutant NP was much less stable than the wild-type NP when enzyme solutions were dialyzed at neutral pH in the presence of [Ca2+] below 10(-3) M. On the other hand, the kinetic thermal stability to irreversible inactivation of mutant NP, when measured in the presence of 0.1 M CaCl2, was found to be increased about 2-fold over that of the wild-type NP. Thus, modulation of enzyme stability by free [Ca2+] in mutant NP correlates with similar findings previously reported for thermolysin. Overall, the results obtained indicate that protein engineering experiments can be used to prepare hybrid proteins on the basis of sequence and function analysis of homologous protein molecules and show the feasibility of engineering metal ion binding sites into proteins.

The overall folding of neutral protease from Bacillus subtilis has been predicted by computer-aided modelling, taking as a basis the known three-dimensional structure of thermolysin. As expected from the 50% similarity of sequence between the two proteins, the structure of B. subtilis protease is similar to that of thermolysin, including the two-domain topology and location of elements of regular secondary structure (helices and strands), whereas specific differences were predicted in loop regions. A protruding and loose loop predicted in B. subtilis has been detected also experimentally by a limited proteolysis approach. Incubation of B. subtilis protease at pH 9.0 for 24 h at room temperature with trypsin at 20: 1 ratio (by mass) leads to a specific and almost quantitative fission of the Arg214-Asn215 peptide bond located in a highly exposed, and thus probably flexible, loop of the protease. On the other hand, thermolysin was completely resistant to tryptic hydrolysis when reacted under identical conditions. The 'nicked' B. subtilis protease can be isolated by gel filtration chromatography at neutral pH, whereas the two constituting fragments 1 -214 and 21 5 -300 are separated under protein-denaturing conditions.Overall, these results indicate that the limited proteolysis approach can pinpoint a peculiar difference in surface structure between the two similar protein molecules of B. subtilis neutral protease and thermolysin and emphasize the potential use of proteolytic enzymes as structural probes of globular proteins. subtilis [lo] are known. These sequence data, obtained by direct protein sequencing or by analyzing the corresponding cDNA, clearly established that these proteases show high degrees of sequence similarities, this being higher among thennostable proteases derived from thermophilic microorganisms, as well as among thermolabile ones from mesophilic sources. Moreover, the crystal structure of thermolysin [I 1 -141 and of the mesophilic neutral protease from B. cereus [15] have been reported. Thus, this ample set of well characterized (thermophilic and mesophilic) neutral proteases constitute an excellent model system for addressing problems of structure/ functionlstability relationships. As a matter of fact, initial studies of protein engineering experiments on neutral proteases by site-directed mutagenesis have already appeared [16, 171. Correspondence to A. Fontana, Department of Organic Chemistry, Via Marzolo 1, 1-35131 Padua, Italy.Abbreviations. Gdn In the ambit of a research program aimed to prepare and study mutants of B. subtilis neutral protease (NP) [17], it was of importance to develop a model of this protease in order to design interesting mutants and to interpret the possible effects of site-specific mutagenesis in terms of conformational details and interactions within the protein molecule. In this paper we report a proposed model of NP predicted on the basis of the known three-dimensional structure of TLN [14] with the aid of a graphics display. As expected from the 50% seq...

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