Mechanism of the binding of Z-L-tryptophan and Z-L-phenylalanine to thermolysin and stromelysin-1 in aqueous solutions

The chemical shift of the carboxylate carbon of Z-tryptophan is increased from 179.85 to 182.82 ppm and 182.87 ppm on binding to thermolysin and stromelysin-1 respectively. The chemical shift of Z-phenylalanine is also increased from 179.5 ppm to 182.9 ppm on binding to thermolysin. From pH studies we conclude that the pK(a) of the inhibitor carboxylate group is lowered by at least 1.5 pK(a) units when it binds to either enzyme. The signal at ~183 ppm is no longer observed when the active site zinc atom of thermolysin or stromelysin-1 is replaced by cobalt. We estimate that the distance of the carboxylate carbon of Z-[1-(13)C]-L-tryptophan is ≤3.71? from the active site cobalt atom of thermolysin. We conclude that the side chain of Z-[1-(13)C]-L-tryptophan is not bound in the S(2)' subsite of thermolysin. As the chemical shifts of the carboxylate carbons of the bound inhibitors are all ~183 ppm we conclude that they are all bound in a similar way most probably with the inhibitor carboxylate group directly coordinated to the active site zinc atom. Our spectrophotometric results confirm that the active site zinc atom is tetrahedrally coordinated when the inhibitors Z-tryptophan or Z-phenylalanine are bound to thermolysin.     

Mechanism of the binding of Z-L-tryptophan and Z-L-phenylalanine to thermolysin and stromelysin-1 in aqueous solutions

The chemical shift of the carboxylate carbon of Z-tryptophan is increased from 179.85 to 182.82 ppm and 182.87 ppm on binding to thermolysin and stromelysin-1 respectively. The chemical shift of Z-phenylalanine is also increased from 179.5 ppm to 182.9 ppm on binding to thermolysin. From pH studies we conclude that the pK_a of the inhibitor carboxylate group is lowered by at least 1.5 pK_a units when it binds to either enzyme. The signal at ~ 183 ppm is no longer observed when the active site zinc atom of thermolysin or stromelysin-1 is replaced by cobalt. We estimate that the distance of the carboxylate carbon of Z-[1-¹³C]-L-tryptophan is ≤ 3.71 Å from the active site cobalt atom of thermolysin. We conclude that the side chain of Z-[1-¹³C]-L-tryptophan is not bound in the S₂′ subsite of thermolysin. As the chemical shifts of the carboxylate carbons of the bound inhibitors are all ~ 183 ppm we conclude that they are all bound in a similar way most probably with the inhibitor carboxylate group directly coordinated to the active site zinc atom. Our spectrophotometric results confirm that the active site zinc atom is tetrahedrally coordinated when the inhibitors Z-tryptophan or Z-phenylalanine are bound to thermolysin.     

Engineering of the pH-dependence of thermolysin activity as examined by site-directed mutagenesis of Asn112 located at the active site of thermolysin

Asn112 is located at the active site of thermolysin, 5-8 A from the catalytic Zn2+ and catalytic residues Glu143 and His231. When Asn112 was replaced with Ala, Asp, Glu, Lys, His, and Arg by site-directed mutagenesis, the mutant enzymes N112D and N112E, in which Asn112 is replaced with Asp and Glu, respectively, were secreted as an active form into Escherichia coli culture medium, while the other four were not. In the hydrolysis of a neutral substrate N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide, the kcat/Km values of N112D and N112E exhibited bell-shaped pH-dependence, as did the wild-type thermolysin (WT). The acidic pKa of N112D was 5.7 +/- 0.1, higher by 0.4 +/- 0.2 units than that of WT, suggesting that the introduced negative charge suppressed the protonation of Glu143 or Zn2+-OH. In the hydrolysis of a negatively charged substrate, N-carbobenzoxy-l-Asp-l-Phe methyl ester (ZDFM), the pH-dependence of kcat/Km of the mutants decreased with increase in pH from 5.5 to 8.5, while that of WT was bell-shaped. This difference might be explained by the electrostatic repulsion between the introduced Asp/Glu and ZDFM, suggesting that introducing ionizing residues into the active site of thermolysin might be an effective means of modifying its pH-activity profile.     

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.     

The primary structure of Bacillus cereus neutral proteinase and comparison with thermolysin and Bacillus subtilis neutral proteinase

The complete amino-acid sequence of a neutral proteinase, produced by Bacillus cereus, was determined by protein sequencing. The neutral proteinase consists of 317 amino-acid residues. The primary structure is 70% homologous to thermolysin, a thermostable neutral proteinase and 45% homologous to Bacillus subtilis neutral proteinase. The zinc-binding site and the hydrophobic pocket of the active site are highly similar in all three proteinases. B. cereus neutral proteinase which is 20 degrees C less thermostable (60 degrees C) than thermolysin (80 degrees C) shows only minor differences in calcium binding sites and salt bridges compared to thermolysin (known from its X-ray diffraction analysis), whereas B. subtilis neutral proteinase (50 degrees C) differs considerably. Therefore it was assumed that the difference in thermostability between B. cereus neutral proteinase and thermolysin is not caused by different metal binding properties, or differences in the active site, but by changes within the rest of the molecule. Calculation of secondary structure potentials according to Chou & Fasman, hydrophobicity and bulkiness of the different structural elements and preferred cold----hot amino-acid residue exchanges indicated, that the thermostability of thermolysin compared to B. cereus neutral proteinase is caused by small effects contributed by numerous amino-acid exchanges distributed over the whole molecule, resulting in increased hydrophobicity of beta-pleated sheet and higher bulkiness of alpha-helical regions.     

Stromelysin-1: three-dimensional structure of the inhibited catalytic domain and of the C-truncated proenzyme.

The proteolytic enzyme stromelysin-1 is a member of the family of matrix metalloproteinases and is believed to play a role in pathological conditions such as arthritis and tumor invasion. Stromelysin-1 is synthesized as a pro-enzyme that is activated by removal of an N-terminal prodomain. The active enzyme contains a catalytic domain and a C-terminal hemopexin domain believed to participate in macromolecular substrate recognition. We have determined the three-dimensional structures of both a C-truncated form of the proenzyme and an inhibited complex of the catalytic domain by X-ray diffraction analysis. The catalytic core is very similar in the two forms and is similar to the homologous domain in fibroblast and neutrophil collagenases, as well as to the stromelysin structure determined by NMR. The prodomain is a separate folding unit containing three alpha-helices and an extended peptide that lies in the active site of the enzyme. Surprisingly, the amino-to-carboxyl direction of this peptide chain is opposite to that adopted by the inhibitor and by previously reported inhibitors of collagenase. Comparison of the active site of stromelysin with that of thermolysin reveals that most of the residues proposed to play significant roles in the enzymatic mechanism of thermolysin have equivalents in stromelysin, but that three residues implicated in the catalytic mechanism of thermolysin are not represented in stromelysin.     

Conformational dynamics of free and catalytically active thermolysin are indistinguishable by hydrogen/deuterium exchange mass spectrometry

Conformational dynamics are thought to be a prerequisite for the catalytic activity of enzymes. However, the exact relationship between structural fluctuations and function is not well understood. In this work hydrogen/deuterium exchange (HDX) and electrospray ionization mass spectrometry (ESI-MS) are used for exploring the conformational dynamics of thermolysin. Amide HDX reflects the internal mobility of proteins; regions that undergo frequent unfolding-refolding show faster exchange than segments that are highly stable. Thermolysin is a zinc protease with an active site that is located between two lobes. Substrate turnover is associated with hinge bending that leads to a closed conformation. Product release regenerates the open form, such that steady-state catalysis involves a continuous closing/opening cycle. HDX/ESI-MS with proteolytic peptide mapping in the absence of substrate shows that elements in the periphery of the two lobes are most mobile. A comparison with previous X-ray data suggests that these peripheral regions undergo quite pronounced structural changes during the catalytic cycle. In contrast, active site residues exhibit only a moderate degree of backbone flexibility, and the central zinc appears to be in a fairly rigid environment. The presence of both rigid and moderately flexible elements in the active site may reflect a carefully tuned balance that is required for function. Interestingly, the HDX behavior of catalytically active thermolysin is indistinguishable from that of the free enzyme. This result is consistent with the view that catalytically relevant motions preexist in the resting state and that enzyme function can only be performed within the limitations given by the intrinsic dynamics of the protein. The data presented in this work indicate the prevalence of stochastic elements in the function of thermolysin, rather than supporting a deterministic mechanism.     

Relationship between the inhibitory potencies of thiorphan and retrothiorphan enantiomers on thermolysin and neutral endopeptidase 24.11 and their interactions with the thermolysin active site by computer modelling

The inhibitory potency of separate enantiomers of thiorphan and retrothiorphan has shown that several particularities of the active site of thermolysin are also present in the neutral endopeptidase 24.11, "enkephalinase", such as its ability: i) to recognize a retroamide bond as well as a standard amide bond, ii) to interact similarly with residues in P1' position of either R or S configuration in the thiorphan series but contrastingly to discriminate between the R and S isomers in the retrothiorphan series. These four inhibitors were modellized in the thermolysin active site and their spatial arrangement compared with that of a thiol inhibitor co-crystallized with thermolysin. In all cases, the essential interactions involved in the stabilization of the bound inhibitor were conserved. However, the bound (R) retrothiorphan displayed unfavorable intramolecular contacts, accounting for its lower inhibitory potency for the two metallopeptidases.     

The protein-protein interactions between SMPI and thermolysin studied by molecular dynamics and MM/PBSA calculations

Thermolysin is a zinc-metalloendopeptidase secreted by the gram-positive thermophilic bacterium Bacillus thermoproteolyticus. Thermolysin belongs to the gluzinicin family of enzymes, which is selectively inhibited by Steptomyces metalloproteinase inhibitor (SMPI). Very little is known about the interaction between SMPI and thermolysin. Knowledge about the protein-protein interactions is very important for designing new thermolysin inhibitors with possible industrial or pharmaceutical applications. In the present study, two binding modes between SMPI and thermolysin were studied by 2300 picoseconds (ps) of comparative molecular dynamics (MD) simulations and calculation of the free energy of binding using the molecular mechanics-Poisson-Boltmann surface area (MM/PBSA) method. One of the positions, the 'horizontal arrow head docking' (HAHD) was similar to the previously proposed binding mode by Tate et al. (Tate, S., Ohno, A., Seeram, S. S., Hiraga, K., Oda, K., and Kainosho, M. J. Mol. Biol. 282, 435-446 (1998)). The other position, the 'vertical arrow head docking' (VAHD) was obtained by a manual docking guided by the shape and charge distribution of SMPI and the binding pocket of thermolysin. The calculations showed that SMPI had stronger interactions with thermolysin in the VAHD than in the HAHD complex, and the VAHD complex was considered more realistic than the HAHD complex. SMPI interacted with thermolysin not only at the active site but had auxiliary binding sites contributing to proper interactions. The VAHD complex can be used for designing small molecule inhibitors mimicking the SMPI-thermolysin binding interfaces.     

Esterase activity of zinc neutral proteases.

The hydrolysis of a series of depsipeptides demonstrates that the zinc neutral endopeptidases of bacteria are active esterases. Esters such as BzGly-OPhe-Ala, BzGly-OLeu-Ala, and FA-Gly-OLeu-NH2 are hydrolyzed at rates three- to eightfold slower than are their exact peptide analogues, when hydrolyzed by thermolysin, Bacillus subtilis neutral protease and the neutral protease from Aeromonas proteolytica. Ester hydrolysis by zinc neutral proteases follows the characteristic preference for hydrophobic amino acids adjacent to the site of cleavage, discerned from the hydrolysis of peptide substrates. Removal of zinc from thermolysin abolishes the esterase activity of the native enzyme. Among the metals examined, only Co2+ and Zn2+ restore esterase activity to any significant extent, Co2+ restoring 50% and Zn2+ 100% of the native thermolysin activity. The hydrolysis of esters and peptides by thermolysin does not differ with respect to either the binding or catalytic steps. Substrate specificity, pH-rate profiles, inhibitor, and deuterium isotope effects are identical for both types of substrates.     

Structural analysis of silanediols as transition-state-analogue inhibitors of the benchmark metalloprotease thermolysin

Dialkylsilanediols have been found to be an effective functional group for the design of active-site-directed protease inhibitors, including aspartic (HIV protease) and metallo (ACE and thermolysin) proteases. The use of silanediols is predicated on its resemblance to the hydrated carbonyl transition-state structure of amide hydrolysis. This concept has been tested by replacing the presumed tetrahedral carbon of a thermolysin substrate with a silanediol group, resulting in an inhibitor with an inhibition constant K(i) = 40 nM. The structure of the silanediol bound to the active site of thermolysin was found to have a conformation very similar to that of a corresponding phosphonamidate inhibitor (K(i) = 10 nM). In both cases, a single oxygen is within bonding distance to the active-site zinc ion, mimicking the presumed tetrahedral transition state. There are binding differences that appear to be related to the presence or absence of protons on the oxygens attached to the silicon or phosphorus. This is the first crystal structure of an organosilane bound to the active site of a protease.     

Thermolysin activation mutants with changes in the fusogenic region of an influenza virus hemagglutinin.

Influenza virus A/seal/Mass/1/80 (H7N7) mutants were obtained; the hemagglutinins (HAs) of the mutants were not activated by trypsin, as in the wild-type virus, but by thermolysin. The mutants grew efficiently under multiple replication cycle conditions and formed plaques in chicken embryo cells only when thermolysin was added to the culture medium. They exhibited hemolytic activity and induced protective immunity in chickens after an asymptomatic course of infection. Nucleotide sequencing of the HA gene and direct amino acid sequencing showed that insertion of a single leucine into the fusion peptide of the HA2 chain close to the cleavage site and a shift of the cleavage site toward the C terminus by one amino acid were responsible for the changes in the biological properties of the thermolysin activation mutants. Revertants could be obtained when trypsin or trypsin-like endoproteases were present in the virus-producing system.     

Primary structure homologies between two zinc metallopeptidases, the neutral endopeptidase 24.11 ("enkephalinase") and thermolysin, through clustering analysis

Analogies in the sequences of two related zinc metallopeptidases, the bacterial thermolysin (316 amino acids) and the recently cloned neutral endopeptidase 24.11 ("enkephalinase", 749 amino acids), have been demonstrated by a hydrophobic cluster analysis method derived from the Lim theory. Two sequence alignments are proposed for the entire primary structure of thermolysin and the C-terminal part of endopeptidase 24.11. Except for an arginine residue, all the amino acids involved in the active site of thermolysin have been retrieved in both models of endopeptidase 24.11 within conserved clustered structures. The first model is characterized by a deletion of the Ca2+-binding coil present in thermolysin and the second by replacement of this coil by two alpha-helices. In both models an Arg residue can be located in the active site of the neutral endopeptidase.     

The Protein-protein Interactions Between SMPI and Thermolysin Studied by Molecular Dynamics and MM/PBSA Calculations

Abstract

Thermolysin is a zinc-metalloendopeptidase secreted by the gram-positive thermophilic bacterium Bacillus thermoproteolyticus. Thermolysin belongs to the gluzinicin family of enzymes, which is selectively inhibited by Steptomyces metalloproteinase inhibitor (SMPI). Very little is known about the interaction between SMPI and thermolysin. Knowledge about the protein-protein interactions is very important for designing new thermolysin inhibitors with possible industrial or pharmaceutical applications. In the present study, two binding modes between SMPI and thermolysin were studied by 2300 picoseconds (ps) of comparative molecular dynamics (MD) simulations and calculation of the free energy of binding using the molecular mechanics-Poisson-Boltmann surface area (MM/PBSA) method. One of the positions, the ‘horizontal arrow head docking’ (HAHD) was similar to the previously proposed binding mode by Tate et al. (Tate, S., Ohno, A., Seeram, S. S., Hiraga, K., Oda, K., and Kainosho, M. J. Mol. Biol. 282, 435–446 (1998)). The other position, the ‘vertical arrow head docking’ (VAHD) was obtained by a manual docking guided by the shape and charge distribution of SMPI and the binding pocket of thermolysin. The calculations showed that SMPI had stronger interactions with thermolysin in the VAHD than in the HAHD complex, and the VAHD complex was considered more realistic than the HAHD complex. SMPI interacted with thermolysin not only at the active site but had auxiliary binding sites contributing to proper interactions. The VAHD complex can be used for designing small molecule inhibitors mimicking the SMPI-thermolysin binding interfaces.     

Involvement of Val 315 located in the C-terminal region of thermolysin in its expression in Escherichia coli and its thermal stability

Thermolysin is a thermophilic and halophilic zinc metalloproteinase that consists of β-rich N-terminal (residues 1–157) and α-rich C-terminal (residues 158–316) domains. Expression of thermolysin variants truncated from the C-terminus was examined in E. coli culture. The C-terminal Lys316 residue was not significant in the expression, but Val315 was critical. Variants in which Val315 was substituted with fourteen amino acids were prepared. The variants substituted with hydrophobic amino acids such as Leu and Ile were almost the same as wild-type thermolysin (WT) in the expression amount, α-helix content, and stability. Variants with charged (Asp, Glu, Lys, and Arg), bulky (Trp), or small (Gly) amino acids were lower in these characteristics than WT. All variants exhibited considerably high activities (50–100% of WT) in hydrolyzing protein and peptide substrates. The expression amount, helix content, and stability of variants showed good correlation with hydropathy indexes of the amino acids substituted for Val315. Crystallographic study of thermolysin has indicated that V315 is a member of the C-terminal hydrophobic cluster. The results obtained in the present study indicate that stabilization of the cluster increases thermolysin stability and that the variants with higher stability are expressed more in the culture. Although thermolysin activity was not severely affected by the variation at position 315, the stability and specificity were modified significantly, suggesting the long-range interaction between the C-terminal region and active site.     

A new class of potent reversible inhibitors of metallo-proteinases: C-terminal thiol-peptides as zinc-coordinating ligands

A number of substrate analogous peptides containing a phosphoramidate, phosphonate ester, hydroxamate, carboxylate or sulfhydryl group are known to be inhibitors of thermolysin and other metalloproteinases. According to the specificity, most of the inhibitors mimic the prime site of the active center. Hitherto, peptidyl derivatives with a thiol group at the C-terminus have not been described. We have synthesized the protected cysteamides Ac-Ala-Ala-CA-SH and Z-Aa1-Aa2-CA-SH (Aa1: Ala, Pro; Aa2: Ala, Leu). The binding of these thiol peptide inhibitors to the metalloproteinases is characterized first by the coordination of the thiolate group of the inhibitor to the catalytic zinc ion and second by the subsite interaction of the peptide ligand in the active site of the enzyme. All peptide derivatives were competitive inhibitors of the zinc metalloproteinase thermolysin. The strongest inhibition was found with Z-Pro-Leu-CA-SH (Ki = 30 microM). Substitution of the N-protecting benzyloxycarbonyl residue towards the acetyl group in the peptide inhibitor, the inhibition constant decreased about 25 times.     

Three-dimensional structure of the elastase of Pseudomonas aeruginosa at 1.5-A resolution

Pseudomonas aeruginosa elastase (PAE) is a zinc metalloprotease with 301 amino acids. We have crystallized and solved the three-dimensional structure of PAE, using data to 1.5-A resolution, and have refined the native molecular structure to R = 0.188. The overall tertiary structure of the PAE molecule is similar to that of thermolysin, with which it shares 28% amino acid sequence identity. Nearly all of the active site residues that might potentially interact with substrates are identical in the two proteins. However, the active site cleft is significantly more "open" in PAE than in thermolysin.     

Effect of a charged residue at the 213th site of thermolysin on the enzymatic activity

Considering the electrostatic potential of active site, four mutants of thermolysin (EC 3.4.24.4) are designed in an attempt to change the optimum pH of the hydrolytic activity toward acidic regions. On the basis of the numerical calculation of the electrostatic potential in the thermolysin molecule, Asp213 is targeted to be replaced by a basic residue, His, Lys, Arg or a neutral one, Asn. The mutant enzymes are produced inBacillus subtilis as a host using the method of site-directed mutagenesis and their optimum pH values for hydrolyzing a synthetic substrate furylacryloyl-Gly-l-Leu-NH₂ are found to be lowered by 0.2–0.4 pH units with reference to the wild type enzyme. The pl shifts of the mutants are evaluated. Neither optimum pH nor pl shift can be explained by the contribution of the pK change only at the mutation site. We find a clear negative correlation between the activities at pH 7.0 and the pI values among the four mutants and wild-type enzyme. It suggests that the contribution of pK shift of other residues must be taken into account in order to explain the activity change. Little change of thermal stability is observed among the mutants and wild type enzymes.     

Effect of Configuration of the Inhibitors on the Mode of Binding to the Enzyme,Thermolysin

Abstract

Preferred conformations of the competitive inhibitors glycyl-L-phenylalanine and glycyl-D-phenylalanine and their mode of binding to thermolysin have been studied. The difference in configuration is shown to affect significantly the mode of binding to thermolysin. Gly-D-Phe prefers to enter the active site in the global minimum conformation whereas Gly-L-Phe may enter in a higher energy conformation. Moreover, D-enantiomer is shown to have a better fit than the L-counterpart in the active site.     

Effects of site-directed mutagenesis of the loop residue of the N-terminal domain Gly117 of thermolysin on its catalytic activity

In the N-terminal domain of thermolysin, two polypeptide strands, Asn112-Ala113-Phe114-Trp115 and Ser118-Gln119-Met120-Val121-Tyr122, are connected by a short loop, Asn116-Gly117, to form an anti-parallel β-sheet. The Asn112-Trp115 strand is located in the active site, while the Ser118-Tyr122 strand and the Asn116-Gly117 loop are located outside the active site. In this study, we explored the catalytic role of Gly117 by site-directed mutagenesis. Five variants, G117A (Gly117 is replaced by Ala), G117D, G117E, G117K, and G117R, were produced by co-expressing in Escherichia coli the mature and pro domains as independent polypeptides. The production levels were in the order G117E > wild type > G117K, G117R > G117D. G117A was hardly produced. This result is in contrast to our previous one that all 72 active-site thermolysin variants were produced at the similar levels whether they retained activity or not (M. Kusano et al. J. Biochem., 145, 103-113 (2009)). G117E exhibited lower activity in the hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide and higher activity in the hydrolysis of N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester than the wild-type thermolysin. G117K and G117R exhibited considerably reduced activities. This suggests that Gly117 plays an important role in the activity and stability of thermolysin, presumably by affecting the geometries of the Asn112-Trp115 and Ser118-Tyr122 strands.