Effects of cobalt-substitution of the active zinc ion in thermolysin on its activity and active-site microenvironment.

Thermolysin is remarkably activated in the presence of high concentrations (1-5 M) of neutral salts [Inouye, K. (1992) J. Biochem. 112, 335-340]. The activity is enhanced 13-15 times with 4 M NaCl at pH 7.0 and 25 degrees C. Substitution of the active site zinc with other transition metals alters the activity of thermolysin [Holmquist, B. and Vallee, B.L. (1974) J. Biol. Chem. 249, 4601-4607]. Cobalt is the most effective among the transition metals and doubles the activity toward N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide. In this study, the effect of NaCl on the activity of cobalt-substituted thermolysin was examined. Cobalt-substituted thermolysin, with 2.8-fold increased activity compared with the native enzyme, is further activated by the addition of NaCl in an exponential fashion, and the activity is enhanced 13-15 times at 4 M NaCl. The effects of cobalt-substitution and the addition of salt are independent of each other. The activity of cobalt-substituted thermolysin, expressed as k(cat)/K(m), is pH-dependent and controlled by at least two ionizing residues with pK(a) values of 6.0 and 7.8, the acidic pK(a) being slightly higher compared to 5.6 of the native enzyme. These pK(a) values remain constant in the presence of 4 M NaCl, indicating that the electrostatic environment of cobalt-substituted thermolysin is more stable than that of the native enzyme, the acidic pK(a) of which shifts remarkably from 5.6 to 6.7 at 4 M NaCl. Zincov, a competitive inhibitor, binds more tightly to the cobalt-substituted than to native thermolysin at pH 4.9-9.0, probably because of its preference for cobalt in the fivefold coordination. The cobalt substitution has been shown to be a favorable tool with which to explore the active-site microenvironment of thermolysin.     

Effects of site-directed mutagenesis of the surface residues Gln128 and Gln225 of thermolysin on its catalytic activity

Thermolysin is remarkably activated and stabilized by neutral salts with varying degrees depending on salt species, and particular surface residues are thought to be especially important in its activity and stability [Inouye, K. (1992) J. Biochem. 112, 335-340; Inouye, K. et al. (1998) Biochim. Biophys. Acta 1388, 209-214]. In this study, we examined the mutational effects of the surface residues of thermolysin. Gln128 and Gln225 were selected as the residues to be mutated because they are located on the surface loop and close to but not in the active site (23.5 and 15.8 A far from the active site zinc ion, respectively) and fully solvent accessible. Nine single mutants [Q128K (Gln128 is replaced with Lys), Q128E, Q128A, Q225K, Q225R, Q225E, Q225D, Q225A and Q225V] were constructed by site-directed mutagenesis. Mutational changes in catalytic activity were found only in the mutant thermolysins having a hydrophobic residue at the position 225 (Q225A and Q225V). In the hydrolysis of a neutral substrate N-[3-(2-furyl)acryloyl]-glycyl-l-leucine amide (FAGLA), the alkaline pK(a) value of Q225A is 8.48 +/- 0.04, being higher by 0.42 +/- 0.07 units than that of the wild-type thermolysin. The k(cat)/K(m) value of the wild-type enzyme is enhanced 14 times with 4 M NaCl, and those of Q225A and Q225V are enhanced 10 and 19 times, respectively. In the hydrolysis of a negatively charged substrate N-carbobenzoxy-l-aspartyl-l-phenylalanine methyl ester (ZDFM), unlike FAGLA, the initial velocities of Q225A and Q225V decreased to 30 and 50% of that of the wild-type enzyme, respectively. Their thermal stability is similar to that of the wild-type enzyme. These findings indicate that even a single mutation at the thermolysin surface induces changes in the electrostatic environment in the active site and affects the activity. Thus, site-directed mutagenesis of surface residues of thermolysin, including apparently thermodynamically unfavorable introduction of hydrophobic residues, should be explored to improve its activity and stability.     

Substrate-dependent activation of thermolysin by salt

Salt-activation of thermolysin was examined using a positively charged fluorescent substrate, (7-methoxycoumarin-4-yl)acetyl-L-Pro-L-Leu-Gly-L-Leu-[N(3)-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]-L-Ala-L-Arg-NH(2) [MOCAc-PLGL(Dpa)AR]. Thermolysin activity increased in a biphasic exponential fashion and was 40 times higher in the presence of 4 M NaCl than in its absence. The degree of activation at X M NaCl was expressed as 4.7(x) when [NaCl](o) < 0.5 M and 2.3(x) when [NaCl](o) > 0.5 M respectively.     

Kinetic analysis of the activation-and-inhibition dual effects of cobalt ion on thermolysin activity

Thermolysin activity as well as its stability is remarkably enhanced by high concentration of neutral salts consisting of Na(+), K(+), Cl(-) and Br(-) in the synthesis and hydrolysis of N-carbobenzoxy-L-aspertyl-L-phenylalanine methyl ester and hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide (FAGLA) [Inouye, K. (1992) J. Biochem. 112, 335-340]. However, effect of divalent salts on thermolysin activity has not been investigated systematically. In this study, effect of Co(2+) ion on thermolysin activity in the hydrolysis of FAGLA was examined. Thermolysin activity increased 3-4 times with increasing the Co(2+) concentration to 2 mM, but the enhanced activity was considerably reduced with higher Co(2+) concentration (2-18 mM). The activation-and-inhibition dual effects of Co(2+) ion were analysed kinetically. Release of the catalytic Zn(2+) ion from thermolysin, concomitantly occurred with the Co(2+)-dependent activation, was measured with a Zn(2+)-specific fluorescent probe. This indicates that the activation is caused by substituting Co(2+) ion for the catalytic Zn(2+) ion. Meanwhile, the Co(2+)-dependent activation was inhibited competitively by Zn(2+) ion (0.1-1.0 muM) added, similarly to that it is inhibited by higher concentration of Co(2+) ion. These lines of evidence provide a strategy for regulating thermolysin activity with Co(2+) and Zn(2+) ions.     

Effects of introducing negative charges into the molecular surface of thermolysin by site-directed mutagenesis on its activity and stability

Thermolysin is remarkably activated and stabilized by neutral salts, and surface charges are suggested important in its activity and stability. The effects of introducing negative charge into the molecular surface on its activity and stability are described. Seven serine residues were selected, and each of them was changed for aspartate by site-directed mutagenesis in a thermolysin mutant. In the hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-l-leucine amide, the k(cat)/K(m) values of all mutants were almost similar to that of the wild-type enzyme (WT). However, those of six out of seven mutants were enhanced 17-19 times with 4 M NaCl, being slightly higher than WT. The remaining casein-hydrolyzing activities of the S53D and S65D mutants (Ser53 and Ser65 are replaced with Asp, respectively) after 30-min incubation with 10 mM CaCl(2) at 85 degrees C were 78 and 63%, being higher than those of WT (51%) and the other mutants (35-53%). S53D was stabilized with increase in the enthalpy change of activation for thermal inactivation while S65D was with decrease in the entropy change of activation. The stability of WT was enhanced by CaCl(2) and reached the level of S53D and S65D at 100 mM, suggesting that S53D and S65D might be stabilized by reinforcement of the Ca(2+)-binding structures.     

Effects of salts on the interaction of 8-anilinonaphthalene 1-sulphonate and thermolysin

Neutral salts activate and stabilize thermolysin. In this study, to explore the mechanism, we analyzed the interaction of 8-anilinonaphthalene 1-sulphonate (ANS) and thermolysin by ANS fluorescence. At pH 7.5, the fluorescence of ANS increased and blue-shifted with increasing concentrations (0–2.0?μM) of thermolysin, indicating that the anilinonaphthalene group of ANS binds with thermolysin through hydrophobic interaction. ANS did not alter thermolysin activity. The dissociation constants (K_d) of the complex between ANS and thermolysin was 33?±?2?μM at 0?M NaCl at pH 7.5, decreased with increasing NaCl concentrations, and reached 9?±?3?μM at 4?M NaCl. The K_d values were not varied (31?34?μM) in a pH range of 5.5?8.5. This suggests that at high NaCl concentrations, Na⁺ and/or Cl^– ions bind with thermolysin and affect the binding of ANS with thermolysin. Our results also suggest that the activation and stabilization of thermolysin by NaCl are partially brought about by the binding of Na⁺ and/or Cl^– ions with thermolysin.     

Hydrolysis of β-lactoglobulin by thermolysin and pepsin under high hydrostatic pressure

Hydrolysis of β-lactoglobulin with thermolysin and pepsin at pressures ranging between 0.1 and 350 MPa showed a significant increase of cleavage rates. Pressure-induced changes of susceptibility to hydrolysis of β-lactoglobulin proteolytic sites were also observed. The pressure, raised to 200 MPa, accelerates the hydrolysis of β-lactoglobulin by thermolysin and changes obtained peptide profiles. Initially, higher pressure makes the N-terminal, and to a smaller extent, C-terminal peptide fragments of β-lactoglobulin molecule, more susceptible to removal by thermolysin. This indicates combined influence of pressure-induced thermolysin activation and partial unfolding of β-lactoglobulin by compression at neutral pHs. The rates of hydrolysis of β-lactoglobulin by pepsin (negligible at 0.1 MPa) are increased considerably with pressure up to 300 MPa. The Susceptibility of β-lactoglobulin proteolytic sites to peptic cleavage remains constant over all the studied pressure range. The lack of significant qualitative changes in the peptic peptide profiles produced at different pressures and at clearly pressure-dependent rates points to negative reaction volume changes as the major factor in peptic hydrolysis of β-lactoglobulin under high pressure. Thus the β-lactoglobulin molecule resists pressure-induced unfolding in acid pHs and yields to it in neutral pHs. © 1995 John Wiley & Sons, Inc.     

Equilibrium study on the binding between thermolysin and Streptomyces metalloprotease inhibitor, talopeptin (MKI)

The binding between thermolysin and its specific inhibitor, talopeptin (MKI), was found to show a fluorescence increase when excited at 280 nm and 295 nm, and a difference spectrum characterized by two peaks at 294 nm and 285 nm with a shoulder around 278 nm, indicating a microenvironmental change in tryptophan residue(s) of thermolysin and/or talopeptin. The inhibitor constant of talopeptin against thermolysin, Ki, was determined over the pH range 5-9 from the inhibition of the enzyme activity towards 3-(2-furylacryloyl)-glycyl-L-leucine amide (FAGLA) as a substrate. The dissociation constant of thermolysin-talopeptin complex, Kd, determined directly from fluorometric titration was in good agreement with the inhibitor constant, Ki, between pH 6 and 8.5. The pH dependence of Ki and Kd suggested that at least two ionizable groups of thermolysin in their protonated forms are essential for the binding between thermolysin and talopeptin. The temperature dependence of K1 at pH 5.5 indicated that the binding is largely exothermic (delta H degree = -12 kcal/mol) and essentially enthalpy-driven.     

Effects of dimethyl sulfoxide, temperature, and sodium chloride on the activity of human matrix metalloproteinase 7 (matrilysin)

Effects of dimethyl sulfoxide (DMSO), temperature, and sodium chloride on the matrilysin-catalyzed hydrolysis of (7-methoxycoumarin-4-yl)acetyl-L-Pro-L-Leu-Gly-L-Leu-[N(3)-(2, 4-dinitrophenyl)-L-2,3-diamino-propionyl]-L-Ala-L-Arg-NH(2) [MOCAc-PLGL(Dpa)AR] were examined. DMSO inhibited the matrilysin activity competitively with the inhibitor constant (K(i)) of 0. 59+/-0.04 M, and the binding between them was endothermic and entropy-driven. The binding of matrilysin with MOCAc-PLGL(Dpa)AR was also found to be entropy-driven. The matrilysin activity was increased in a biphasic exponential fashion with increasing concentration of NaCl, and was 5.3 times higher in the presence of 4 M NaCl than that in its absence. The first and second phases were separated at 0.5 M NaCl, and the activation at x M NaCl compared with the activity in the absence of NaCl was expressed as 2.1(x) at [NaCl] < 0.5 M and 1.4(x) at [NaCl] > 0.5 M. The activation was brought about solely through a decrease in the Michaelis constant (K(m)), and the catalytic constant (k(cat)) was not much altered. This suggests that the decrease in the electrostatic interaction and the increase in the hydrophobic interaction between matrilysin and the substrate might enhance the enzyme activity by reducing the K(m) value.     

Characterization of Gly-D-Phe, Gly-L-Leu, and D-Phe as affinity ligands to thermolysin

In this study, glycyl-D-phenylalanine (Gly-D-Phe), glycyl-L-leucine (Gly-L-Leu), and D-phenylalanine (D-Phe) were characterized for their abilities as affinity ligands to thermolysin. Each of the ligands was immobilized to the resin. The optimum pH for adsorption of thermolysin is 5.0-6.0 for each of the ligands. By the affinity column chromatography in which 2mg thermolysin was applied onto 4 ml volume of the resins at pH 5.5, the adsorption ratios based on casein hydrolysis activity were 100% for each of the ligands. However, the adsorption ratios of the resins containing Gly-L-Leu and D-Phe, unlike that of Gly-D-Phe, were progressively decreased with increasing the amounts of thermolysin applied to the column. Measurement of adsorption isotherms showed that the association constant to thermolysin at pH 5.5 of the resins containing Gly-D-Phe was (3.3+/-0.8)x10(5)M(-1), while those of Gly-L-Leu and D-Phe were approximately ten times less. This result is coincident with the observations of performances in affinity column chromatography. On the other hand, maximum thermolysin binding capacities were almost the same among the resins examined. These results indicate that Gly-D-Phe is more suitable than Gly-L-Leu and D-Phe as an affinity ligand for purification of thermolysin.     

Comparison of dipeptidyl carboxypeptidase and endopeptidase activities in the three enkephalin-hydrolysing metallopeptidases: "angiotensin-converting enzyme", thermolysin and "enkephalinase"

Angiotensin-converting enzyme (ACE), thermolysin and "enkephalinase", three metallopeptidases cleaving the Gly3-Phe4 amide bond of enkephalins, were compared regarding substrate specificity and effects of butanedione, an arginyl-directed reagent. The hydrolysis of enkephalins and analogues was more affected by the nature of P1 and P2 residues in the case of thermolysin than in those of ACE or "enkephalinase"; amidation of the C-terminal carboxylate decreased drastically the hydrolysis by ACE but only marginally by thermolysin and the effect was intermediate for "enkephalinase". With adequate model substrates, the ratio of dipeptidylcarboxypeptidase to tripeptidylcaroxypeptidase (endopeptidase) activities were of 25 for ACE, 3 for "enkephalinase" and only 0.3 for thermolysin. Finally a butanedione treatment increased thermolysin activity, but abolished ACE activity; it reduced "enkephalinase" activity by 80% when measured with a free C-terminal carboxylate enkephalin analogue but only slightly with the corresponding amidated derivative. A critical role of an Arg residue in ACE and, to a lesser extent, in "enkephalinase" (but not in thermolysin) is suggested to be responsible for the preferential dipeptidylcarboxypeptidase activity of these two enzymes.     

Inhibitory effects of alcohols on thermolysin activity as examined using a fluorescent substrate

Alcohols inhibit the thermolysin-catalyzed hydrolysis of N-[3-(2-furyl)acryloyl]-Gly-L-Leu-NH(2) and decrease the NaCl-induced activation of thermolysin in a concentration-dependent manner [K. Inouye et al. (1997) J. Biochem. 122, 358-364]. In this study, the inhibitory effects of alcohols on thermolysin activity were examined in detail using 10 different alcohols and a fluorescent substrate, (7-methoxycoumarin-4-yl) acetyl-L-Pro-L-Leu-Gly-L-Leu-[N(3)-(2,4-dinitrophenyl)-L-2,3-diamino-propionyl]-L-Ala-L-Arg-NH(2). The inhibition by all alcohols examined is completely reversible, and thermolysin activity is recovered by dilution. The inhibitor constants (K(i)) are in the range of 35-430 mM, and the order of the inhibitory effect is 1-pentanol, 1-propanol, 2-butanol, 2-methyl-1-propanol > 1-butanol > 2-propanol > ethanol, tert-amyl alcohol > tert-butyl alcohol > methanol. Linear and secondary alcohols whose mains chains consist of more than 3 carbons inhibit thermolysin effectively. Thermolysin activity is decreased by decreasing the dielectric constant, D, of the reaction medium containing the alcohol, and the decrease depending on the D value was almost the same manner for all alcohols except methanol, tert-butyl alcohol, and tert-amyl alcohol. Alcohols may inhibit thermolysin activity both by binding to the active site, most possibly to the S1' subsite, of thermolysin and by altering the electrostatic and hydrophobic environment around the thermolysin molecule.     

Autolysis of thermolysin. Isolation and characterization of a folded three-fragment complex

Incubation of the neutral metalloendopeptidase thermolysin at pH 7.2 in the presence of EDTA and/or low concentrations of calcium ions produces fast enzyme inactivation as a result of autolysis. The 'nicked' protein is a folded species composed of three tightly associated protein fragments. Dissociation of this complex can be achieved under denaturing conditions, such as gel filtration on a column equilibrated with 5 M guanidine hydrochloride or reverse-phase high-performance liquid chromatography (HPLC) at acidic pH. The positions of the peptide bond cleavages were defined by isolation of the individual fragments by HPLC and their characterization by amino acid analysis after acid hydrolysis, end-group determination and partial amino acid sequencing. The results of these analyses indicated that the nicked protein is composed of fragments 1-196, 197-204 and 205-316 and thus that the corresponding sites of limited proteolysis occur at the polypeptide chain loop involved in the binding of Ca(4) in native thermolysin [Matthews, B. W., Weaver, L. H. and Kester, W. R. (1974) J. Biol. Chem. 249, 8030-8044]. The overall conformational properties of nicked thermolysin are quite similar to those of the intact protein, as judged by spectroscopic measurements and by the fact that rabbit antibodies against native thermolysin recognize and precipitate the nicked protein in immunodiffusion assays. The nicked protein was much less stable to heat and unfolding agents than intact thermolysin. These results contribute to a better knowledge of the molecular mechanism of stabilization of native thermolysin by the four bound calcium ions and demonstrate that the function of Ca(4) is to stabilize the loop 190-205 on the surface of the molecule against autolysis.     

Effect of electron withdrawing substituents on substrate hydrolysis by and inhibition of rat neutral endopeptidase 24.11 (enkephalinase) and thermolysin

A series of N-acylphenylalanylglycine dipeptides were synthesized and examined as substrates for neutral endopeptidase 24.11 (NEP) and thermolysin. Those N-acyl dipeptides containing an N-acyl group derived from an acid whose pKa is below 3.5 were considerably more reactive with both enzymes than those peptides containing an N-acyl group derived from an acid whose pKa is above 4. The data are interpreted to suggest that electron withdrawal at the scissile bond increases kappa cat for both NEP and thermolysin. The pH dependence for inhibition by the dipeptides Phe-Ala, Phe-Gly, and Leu-Ala showed binding dependent upon the basic form of an enzyme residue with a pKa of 7 for NEP and a pKa of 6 for thermolysin. In the case of thermolysin this pKa was decreased to 5.3 in the enzyme-inhibitor complex. When examined as alternate substrate inhibitors of NEP, N-acyl dipeptides showed three distinct profiles for the dependence of Ki on pH. With N-trifluoroacetyl-Phe-Gly as inhibitor, binding is dependent upon the basic form of an enzyme residue with a pKa value of 6.2. N-methoxyacetyl-Phe-Gly inhibition appears pH independent, while N-acetyl-Phe-Gly inhibition is dependent upon the acidic form of an enzyme residue with a pKa of approximately 7. All inhibitions of thermolysin by N-acyl dipeptides exhibit a dependence on the acidic form of an enzyme residue with a pKa of 5.3 to 5.8. These results suggest that with NEP, binding interactions at the active site involve one or more histidine residues while with thermolysin binding involves an active site glutamic acid residue.     

Grafting of a calcium-binding loop of thermolysin to Bacillus subtilis neutral protease

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.     

Immobilization of thermolysin to polyamide nonwoven materials

In the last few years, an increasing number of biotechnological techniques have been applied to the restoration and conservation of works of art, paintings, old maps, and papers or books. Enzymes can solve problems that give restorers difficulties, although for many applications it is not possible to use soluble enzymes; therefore, it is necessary to look for suitable carriers for immobilization. Different methods for covalent immobilization of enzymes to polyamide nonwovens were tested, using thermolysin as an example. Two distinct strategies were pursued: (1). controlled, partial hydrolysis of the polymer and subsequent binding of the enzyme to the released amino and carboxy groups; and (2). attachment of reactive groups directly to the polyamide without disintegrating the polymeric structure (O-alkylation). Different spacers were used for covalent fixation of the enzyme in both cases. The enzyme was fixed to the released amino groups by glutaraldehyde, either with or without a spacer. Either way, active enzyme could be immobilized to the matrix. However, intense treatment caused severe damage to the stability of the nonwoven fabric, and reduced the mechanical strength. Conditions were investigated to conserve the nonwoven fabric structure while obtaining near-maximum immobilized enzyme activity. Immobilization of the enzyme to the released carboxy group after acid hydrolysis was performed using dicyclohexylcarbodiimide. In comparison to the enzyme bound via the amino group, the yield of immobilized enzyme activity was slightly lower when benzidine was taken as spacer and still lower with a 1,6-hexanediamine spacer. O-alkylation performed with dimethylsulfate caused severe damage to the nonwoven fabric structure. Considerably better results were obtained with triethyloxonium tetrafluoroborate. As the spacers 1,6-hexanediamine and adipic acid dihydrazide were used, activation for immobilizing thermolysin was performed with glutaraldehyde, adipimidate, and azide. With the exception of azide, all combinations of spacers and activation reagents gave high yields of immobilized enzyme activity. Thermolysin immobilized by this technique showed a remarkably improved stability with respect to elevated temperature, extreme pH values, and reduced polarity. The nonwoven fabric can be stored for weeks without loss of enzyme activity by washing with distilled water and drying.     

Limited proteolysis of thermolysin by subtilisin: isolation and characterization of a partially active enzyme derivative

Incubation of the neutral metalloendopeptidase thermolysin at pH 9-10 in the presence of 10 mM CaCl2 for 2 days at room temperature with subtilisin at a 50:1 molar ratio leads to a derivative possessing lower (approximately 3%) but intrinsic catalytic activity. This derivative, called thermolysin S, was isolated by gel filtration in approximately 80% yield and then separated from some residual intact thermolysin by an affinity chromatographic step on Sepharose-Gly-D-Phe. It was found that thermolysin S results from a tight association of two polypeptide fragments of apparent Mr of 24000 and 10000. Dissociation of the complex was achieved under strong denaturing conditions, such as gel filtration on a column equilibrated and eluted with 5 M guanidine hydrochloride. The positions of the clip sites were defined by amino acid analysis, end-group determination, and amino acid sequencing of the isolated fragments and shown to lie between Thr-4 and Ser-5, between Thr-224 and Gln-225, and also between Gln-225 and Asp-226. Thermolysin S, which is therefore a stable complex of fragments 5-224(225) and 225(226)-316, shows a shift in optimum pH of about 1 unit toward the acid range with respect to intact thermolysin and a Km essentially unchanged, with furylacryloyl-Gly-Leu-NH2 as substrate. Inhibitors of thermolysin such as ethoxyformic anhydride and Zn2+ ions inactivate also the nicked enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of neutral salts and alcohols on the activity of Streptomyces caespitosus neutral protease

Streptomyces caespitosus neutral protease (ScNP) is one of the smallest metalloproteinase with a molecular mass of 14 kDa. Effects of solvent composition on ScNP activity were examined using a peptide substrate. The k(cat)/K(m) values of ScNP exhibited bell-shaped pH-dependence with the optimal pH of 6.4-7.0 and the pK(a) values of 5.0 +/- 0.1 and 8.3 +/- 0.1. ScNP activity increased in an exponential fashion with increasing [NaCl]. The relative k(cat)/K(m) value at 3.6 M NaCl to that at 0 M NaCl was 3.7, and the degree of the activation at x M NaCl was expressed as 1.2 (x) (x < 2.0) and 1.4(x) (x > 2.0). On the other hand, ScNP activity decreased with increasing concentrations of LiCl, KCl, NaBr, LiBr, KBr and NaClO(4). Alcohols inhibited ScNP activity with the IC(50) values, the concentration required for decreasing the activity at 50% of the maximum, of 0.77-6.54 M. The order of the inhibitory potency was 1-butanol, 2-methyl-1-propanol, 2-methyl-2-butanol > 2-methyl-2-propanol, 2-butanol, 1-propanol > 2-propanol > ethanol > methanol. The activities recovered completely by the dilution of alcohols, suggesting that the ScNP inhibition by alcohols is reversible. These characteristics of ScNP are compared with those of human matrix metalloproteinase 7 and thermolysin.     

Mechanism of thermostability in thermolysin – analysis of subsite S2 mutant enzymes of thermolysin

An aromatic amino acid at position 115 (tryptophan residue; subsite S2) in thermolysin is known to be essential for proteolytic activity of thermolysin. Mutant enzymes substituted by phenylalanine (W115F) and tyrosine (W115Y) at position 115 were expressed at similar levels as the wild type (WT) enzyme in Bacillus subtilis . The thermostability of the W115Y mutant enzyme was equal to that of the WT. However, that of the W115F mutant enzyme was significantly lower than the WT. Enzymatic kcat/Km values of W115F increased to about twice those of the WT, but W115F also seemed to promote increased autodegradation compared with the WT and W115Y enzymes.     

Insights into the catalytic roles of the polypeptide regions in the active site of thermolysin and generation of the thermolysin variants with high activity and stability

The active site of thermolysin is composed of one zinc ion and five polypeptide regions [N-terminal sheet (Asn112-Trp115), alpha-helix 1 (Val139-Thr149), C-terminal loop 1 (Asp150-Gly162), alpha-helix 2 (Ala163-Val176) and C-terminal loop 2 (Gln225-Ser234)]. To explore their catalytic roles, we introduced single amino-acid substitutions into these regions by site-directed mutagenesis and examined their effects on the activity and stability. Seventy variants, in which one of the twelve residues (Ala113, Phe114, Trp115, Asp150, Tyr157, Gly162, Ile168, Ser169, Asp170, Asn227, Val230 and Ser234) was replaced, were produced in Escherichia coli. The hydrolytic activities of thermolysin for N-[3-(2-furyl)acryloyl]-Gly-l-Leu amide (FAGLA) and casein revealed that the N-terminal sheet and alpha-helix 2 were critical in catalysis and the C-terminal loops 1 and 2 were in substrate recognition. Twelve variants were active for both substrates. In the hydrolysis of FAGLA and N-carbobenzoxy-L-Asp-L-Phe methyl ester, the k(cat)/K(m) values of the D150E (in which Asp150 is replaced with Glu) and I168A variants were 2-3 times higher than those of the wild-type (WT) enzyme. Thermal inactivation of thermolysin at 80 degrees C was greatly suppressed with the D150H, D150W, I168A, I168H, N227A, N227H and S234A. The evidence might provide the insights into the activation and stabilization of thermolysin.