The Experts below are selected from a list of 288 Experts worldwide ranked by ideXlab platform
Kuniyo Inouye - One of the best experts on this subject based on the ideXlab platform.
-
Effects of salts on the interaction of 8-anilinonaphthalene 1-sulphonate and Thermolysin.
Bioscience Biotechnology and Biochemistry, 2014Co-Authors: Vimbai Samukange, Kiyoshi Yasukawa, Masayuki Kamo, Kuniyo InouyeAbstract: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 (Kd) 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 Kd 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 wit...
-
involvement of val 315 located in the c terminal region of Thermolysin in its expression in escherichia coli and its thermal stability
Biochimica et Biophysica Acta, 2014Co-Authors: Kenji Kojima, Hiroki Nakata, Kuniyo InouyeAbstract:Abstract 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.
-
Effects of Conversion of the Zinc-Binding Motif Sequence of Thermolysin, HEXXH, to That of Dipeptidyl Peptidase III, HEXXXH, on the Activity and Stability of Thermolysin
Bioscience Biotechnology and Biochemistry, 2013Co-Authors: Evans Menach, Kiyoshi Yasukawa, Yasuhiko Hashida, Kuniyo InouyeAbstract:Most zinc metalloproteinases have the consensus zinc-binding motif sequence HEXXH, in which two histidine residues chelate a catalytic zinc ion. The zinc-binding motif sequence of Thermolysin, H142ELTH146, belongs to this motif sequence, while that of dipeptidyl peptidase III (DPP III), H450ELLGH455, belongs to the motif sequence HEXXXH. In this study, we examined effects of conversion of HEXXH to HEXXXH in Thermolysin on its activity and stability. Thermolysin variants bearing H142ELLGH146 or H142ELTGH146 (designated T145LG and T145TG respectively) were constructed by site-directed mutagenesis and were produced in Escherichia coli cells by co-expressing the mature and pro domains separately. They did not exhibit hydrolyzing activity for casein or N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide, but exhibited binding ability to a substrate analog glycyl-D-phenylalanine (Gly-D-Phe). The apparent denaturing temperatures based on the ellipticity at 222 nm of T145LG and T145TG were 85 ± 1 °C and 86 ± 1 °C resp...
-
effects of site directed mutagenesis in the n terminal domain of Thermolysin on its stabilization
Journal of Biochemistry, 2013Co-Authors: Yuichi Kawasaki, Kiyoshi Yasukawa, Kuniyo InouyeAbstract:Thermolysin [EC 3.4.24.27] is a thermostable neutral metalloproteinase produced in the culture broth of Bacillus thermoproteolyticus (1, 2). It consists of 316 amino acid residues with one zinc ion essential for enzyme activity and four calcium ions required for structural stability (3–6). Based on the structural data (7, 8), it consists of a β-rich N-terminal domain and an α-helical C-terminal domain. The Ca2+-binding sites I, II and IV are located in the C-terminal domain, and the Ca2+-binding site III is located in the N-terminal domain (7, 8) (Fig. 1A). When Thermolysin is incubated with ethylenediamine-N,N,N′,N′-tetraacetic acid (EDTA), all calcium ions are removed from the Thermolysin molecule and autolysis occurs (9–11). Thermolysin catalyses specifically the hydrolysis of peptide bonds containing hydrophobic amino acid residues (12, 13). It is widely used for the peptide bond formation through reverse reaction of hydrolysis, in particular, N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester (ZDFM), the precursor of an artificial sweetener aspartame, from N-carbobenzoxy-L-aspartic acid (ZD) and L-phenylalanine methyl ester (FM) (2, 14). Improvement of its activity and stability and modification of its pH-activity profile are thus still important goals (15). We have reported that high concentrations (1–5 M) of neutral salts increase the activity and stability of Thermolysin (13, 14, 16, 17). Fig. 1 Structure of Thermolysin. (A) Whole structure of the WT. The overall protein structure (ribbon model), the mutated residues (ball and stick model) and zinc and calcium ions (sphere) are shown. (B) Close-up view of the Ca2+-binding site III of WT. The ... Site-directed mutagenesis experiments of Thermolysin (18–26) and Thermolysin-like protease (TLP-ste) [EC 3.4.24.4] (27, 28) have generated a number of variant enzymes with improved activity and/or stability. TLP-ste is a neutral metalloproteinase from Bacillus stearothermophilus. It consists of 319 amino acid residues and differs from Thermolysin at 44 out of 319 residues. (In this study, the amino acid sequence of Thermolysin is adopted for numbering that of TLP-ste.) In TLP-ste, a single mutation Ser65→Pro was first reported to increase stability (27). (In this study, the mutation of a residue, e.g. Ser65 to Pro is designated as Ser65→Pro, and the variant enzyme bearing Ser65→Pro is designated as S65P.) After that, a double mutation Gly8→Cys/Asn60→Cys, designed to introduce a disulphide bridge between the amino acid residues 8 and 60, was reported to increase stability (28). In Thermolysin, we reported that the mutational combination of Gly8→Cys/Asn60→Cys and Ser65→Pro increases stability without affecting activity (20). These results suggest that the N-terminal domain is important for stability in Thermolysin and TLP-ste. However, little is known about the mechanism of stabilization by these mutations. In this study, we examined the effects of the mutations Gly8→Cys/Asn60→Cys and Ser65→Pro on the thermal denaturation and inactivation of Thermolysin. Our results suggest that Gly8→Cys/Asn60→Cys and Ser65→Pro stabilize Thermolysin by improving its affinity to calcium ions.
-
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
Journal of Biochemistry, 2008Co-Authors: Masayuki Kusano, Kiyoshi Yasukawa, Kuniyo InouyeAbstract:: 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.
Brian W Matthews - One of the best experts on this subject based on the ideXlab platform.
-
structural analysis of silanediols as transition state analogue inhibitors of the benchmark metalloprotease Thermolysin
Biochemistry, 2005Co-Authors: Douglas H Juers, Brian W Matthews, Scott Mcn SieburthAbstract: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 Ki = 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 (Ki = 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 firs...
-
structural analysis of zinc substitutions in the active site of Thermolysin
Protein Science, 1995Co-Authors: Debra R Holland, Andrew C Hausrath, Doug Juers, Brian W MatthewsAbstract:Native Thermolysin binds a single catalytically essential zinc ion that is tetrahedrally coordinated by three protein ligands and a water molecule. During catalysis the zinc ligation is thought to change from fourfold to fivefold. Substitution of the active-site zinc with Cd2+, Mn2+, Fe2+, and Co2+ alters the catalytic activity (Holmquist B, Vallee BL, 1974, J Biol Chem 249:4601-4607). Excess zinc inhibits the enzyme. To investigate the structural basis of these changes in activity, we have determined the structures of a series of metal-substituted Thermolysins at 1.7-1.9 A resolution. The structure of the Co(2+)-substituted enzyme is shown to be very similar to that of wild type except that two solvent molecules are liganded to the metal at positions that are thought to be occupied by the two oxygens of the hydrated scissile peptide in the transition state. Thus, the enhanced activity toward some substrates of the cobalt-relative to the zinc-substituted enzyme may be due to enhanced stabilization of the transition state. The ability of Zn2+ and Co2+ to accept tetrahedral coordination in the Michaelis complex, as well as fivefold coordination in the transition state, may also contribute to their effectiveness in catalysis. The Cd(2+)- and Mn(2+)-substituted Thermolysins display conformational changes that disrupt the active site to varying degrees and could explain the associated reduction of activity. The conformational changes involve not only the essential catalytic residue, Glu 143, but also concerted side-chain rotations in the adjacent residues Met 120 and Leu 144. Some of these side-chain movements are similar to adjustments that have been observed previously in association with the "hinge-bending" motion that is presumed to occur during catalysis by the zinc endoproteases. In the presence of excess zinc, a second zinc ion is observed to bind at His 231 within 3.2 A of the zinc bound to native Thermolysin, explaining the inhibitory effect.
-
Structural basis for the action of Thermolysin.
Matrix (Stuttgart Germany). Supplement, 1992Co-Authors: Tronrud De, Roderick Sl, Brian W MatthewsAbstract:: High resolution X-ray crystallography has been used to determine the modes of binding to Thermolysin of a series of different inhibitors including dipeptides, mercaptans, hydroxamates, N-carboxymethyl peptides and phosphonamidates. The interactions displayed by such inhibitors illustrate interactions that are presumed to occur between the enzyme and its substrates during catalysis. The crystallographic analysis, together with model building, suggest a detailed stereochemical mechanism of action for Thermolysin and, by analogy, other zinc proteases such as carboxypeptidase A and the angiotensin converting enzyme. Analysis of a series of phosphonamidates, which are presumed to be transition-state analogues, has shown that chemically similar inhibitors can adopt dissimilar modes of binding. These different configurations provide a rationalization for large differences in the kinetics of binding that are observed for these inhibitors. Experiments with Thermolysin as a test case suggest that knowledge of the three-dimensional structure of an enzyme or receptor will greatly facilitate the rational design of drugs directed at such targets.
Kiyoshi Yasukawa - One of the best experts on this subject based on the ideXlab platform.
-
Effects of salts on the interaction of 8-anilinonaphthalene 1-sulphonate and Thermolysin.
Bioscience Biotechnology and Biochemistry, 2014Co-Authors: Vimbai Samukange, Kiyoshi Yasukawa, Masayuki Kamo, Kuniyo InouyeAbstract: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 (Kd) 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 Kd 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 wit...
-
Effects of Conversion of the Zinc-Binding Motif Sequence of Thermolysin, HEXXH, to That of Dipeptidyl Peptidase III, HEXXXH, on the Activity and Stability of Thermolysin
Bioscience Biotechnology and Biochemistry, 2013Co-Authors: Evans Menach, Kiyoshi Yasukawa, Yasuhiko Hashida, Kuniyo InouyeAbstract:Most zinc metalloproteinases have the consensus zinc-binding motif sequence HEXXH, in which two histidine residues chelate a catalytic zinc ion. The zinc-binding motif sequence of Thermolysin, H142ELTH146, belongs to this motif sequence, while that of dipeptidyl peptidase III (DPP III), H450ELLGH455, belongs to the motif sequence HEXXXH. In this study, we examined effects of conversion of HEXXH to HEXXXH in Thermolysin on its activity and stability. Thermolysin variants bearing H142ELLGH146 or H142ELTGH146 (designated T145LG and T145TG respectively) were constructed by site-directed mutagenesis and were produced in Escherichia coli cells by co-expressing the mature and pro domains separately. They did not exhibit hydrolyzing activity for casein or N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide, but exhibited binding ability to a substrate analog glycyl-D-phenylalanine (Gly-D-Phe). The apparent denaturing temperatures based on the ellipticity at 222 nm of T145LG and T145TG were 85 ± 1 °C and 86 ± 1 °C resp...
-
effects of site directed mutagenesis in the n terminal domain of Thermolysin on its stabilization
Journal of Biochemistry, 2013Co-Authors: Yuichi Kawasaki, Kiyoshi Yasukawa, Kuniyo InouyeAbstract:Thermolysin [EC 3.4.24.27] is a thermostable neutral metalloproteinase produced in the culture broth of Bacillus thermoproteolyticus (1, 2). It consists of 316 amino acid residues with one zinc ion essential for enzyme activity and four calcium ions required for structural stability (3–6). Based on the structural data (7, 8), it consists of a β-rich N-terminal domain and an α-helical C-terminal domain. The Ca2+-binding sites I, II and IV are located in the C-terminal domain, and the Ca2+-binding site III is located in the N-terminal domain (7, 8) (Fig. 1A). When Thermolysin is incubated with ethylenediamine-N,N,N′,N′-tetraacetic acid (EDTA), all calcium ions are removed from the Thermolysin molecule and autolysis occurs (9–11). Thermolysin catalyses specifically the hydrolysis of peptide bonds containing hydrophobic amino acid residues (12, 13). It is widely used for the peptide bond formation through reverse reaction of hydrolysis, in particular, N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester (ZDFM), the precursor of an artificial sweetener aspartame, from N-carbobenzoxy-L-aspartic acid (ZD) and L-phenylalanine methyl ester (FM) (2, 14). Improvement of its activity and stability and modification of its pH-activity profile are thus still important goals (15). We have reported that high concentrations (1–5 M) of neutral salts increase the activity and stability of Thermolysin (13, 14, 16, 17). Fig. 1 Structure of Thermolysin. (A) Whole structure of the WT. The overall protein structure (ribbon model), the mutated residues (ball and stick model) and zinc and calcium ions (sphere) are shown. (B) Close-up view of the Ca2+-binding site III of WT. The ... Site-directed mutagenesis experiments of Thermolysin (18–26) and Thermolysin-like protease (TLP-ste) [EC 3.4.24.4] (27, 28) have generated a number of variant enzymes with improved activity and/or stability. TLP-ste is a neutral metalloproteinase from Bacillus stearothermophilus. It consists of 319 amino acid residues and differs from Thermolysin at 44 out of 319 residues. (In this study, the amino acid sequence of Thermolysin is adopted for numbering that of TLP-ste.) In TLP-ste, a single mutation Ser65→Pro was first reported to increase stability (27). (In this study, the mutation of a residue, e.g. Ser65 to Pro is designated as Ser65→Pro, and the variant enzyme bearing Ser65→Pro is designated as S65P.) After that, a double mutation Gly8→Cys/Asn60→Cys, designed to introduce a disulphide bridge between the amino acid residues 8 and 60, was reported to increase stability (28). In Thermolysin, we reported that the mutational combination of Gly8→Cys/Asn60→Cys and Ser65→Pro increases stability without affecting activity (20). These results suggest that the N-terminal domain is important for stability in Thermolysin and TLP-ste. However, little is known about the mechanism of stabilization by these mutations. In this study, we examined the effects of the mutations Gly8→Cys/Asn60→Cys and Ser65→Pro on the thermal denaturation and inactivation of Thermolysin. Our results suggest that Gly8→Cys/Asn60→Cys and Ser65→Pro stabilize Thermolysin by improving its affinity to calcium ions.
-
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
Journal of Biochemistry, 2008Co-Authors: Masayuki Kusano, Kiyoshi Yasukawa, Kuniyo InouyeAbstract:: 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.
-
a new method for the extracellular production of recombinant Thermolysin by co expressing the mature sequence and pro sequence in escherichia coli
Protein Engineering Design & Selection, 2007Co-Authors: Kiyoshi Yasukawa, Masayuki Kusano, Kuniyo InouyeAbstract:: Thermolysin, a representative zinc metalloproteinase from Bacillus thermoproteolyticus, is synthesized as inactive pre-proenzyme and receives autocatalytic cleavage of the peptide bond linking the pro- and mature sequences. The conventional expression method for recombinant Thermolysin requires the autocatalytic cleavage, so that production of a mutant Thermolysin is affected by its autocatalytic digestion activity. In this study, we have established a new expression method that does not require the autocatalytic cleavage. The mature sequence of Thermolysin containing an NH(2)-terminal pelB leader sequence and the pre-prosequence of Thermolysin were co-expressed constitutively in Escherichia coli as independent polypeptides under the original promoter sequences in the npr gene which encodes Thermolysin. Unlike the conventional expression method, not only the wild-type Thermolysin but also mutant Thermolysins [E143A (Glu143 is replaced with Ala), N112A, N112D, N112E, N112H, N112K and N112R] were produced into the culture medium. The wild-type enzyme expressed in the present method was indistinguishable from that expressed in the conventional method based on autocatalytic cleavage, as assessed by hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide and N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester. The present method should be useful especially for preparation of active-site mutants of Thermolysin, which might have suppressed autocatalytic digestion activity. The results also demonstrate clearly that the covalent linking between the pro- and mature sequences is not necessary for the proper folding of the mature sequence by the propeptide in Thermolysin.
G Venema - One of the best experts on this subject based on the ideXlab platform.
-
the effect of changing the hydrophobic s1 subsite of Thermolysin like proteases on substrate specificity
FEBS Journal, 2001Co-Authors: A De Kreij, G Vriend, G Venema, B Van Den Burg, Oene R Veltman, V G H EijsinkAbstract:The hydrophobic S-1' 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. The mutational effects showed that the wild-type S1' pocket is optimal for binding leucine side chains. Reduction of the size of residue 202 resulted in a higher efficiency towards substrates with Phe in the P-1' position. Rather unexpectedly, the Leu202-->Phe and Leu202-->Tyr mutations, which were expected to decrease the size of the S-1' subsite, resulted in a large increase in activity towards dipeptide substrates with Phe in the P-1' position. This is probably due to the fact that 202Phe and 202Tyr adopt a second possible rotamer that opens up the subsite compared to Leu202, and also favours interactions with the substrate. To validate these results, we constructed variants of Thermolysin with changes in the S-1' subsite. Thermolysin and TLP-ste variants with identical S-1' subsites were highly similar in terms of their preference for Phe vs. Leu in the P-1' position.
-
structural determinants of the stability of Thermolysin like proteinases
Nature Structural & Molecular Biology, 1995Co-Authors: V G H Eijsink, O R Veltman, W Aukema, G Vriend, G VenemaAbstract:Thermolysin is a member of a family of homologous proteinases which differ in their resistance to thermally induced unfolding and subsequent autolytic degradation. Site-directed mutagenesis studies of the Thermolysin-like proteinase (TLP) from Bacillus stearothermophilus (TLP-ste) show that its reduced resistance to thermally induced autolysis, as compared to Thermolysin, is due to only some of the 44 naturally occurring amino-acid differences between them. In fact TLP-ste becomes more resistant than Thermolysin by mutation of just a few of these amino-acids. The crucial differences are all localized to a solvent-exposed region in the N-terminal domain of TLP-ste.
V G H Eijsink - One of the best experts on this subject based on the ideXlab platform.
-
the effect of changing the hydrophobic s1 subsite of Thermolysin like proteases on substrate specificity
FEBS Journal, 2001Co-Authors: A De Kreij, G Vriend, G Venema, B Van Den Burg, Oene R Veltman, V G H EijsinkAbstract:The hydrophobic S-1' 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. The mutational effects showed that the wild-type S1' pocket is optimal for binding leucine side chains. Reduction of the size of residue 202 resulted in a higher efficiency towards substrates with Phe in the P-1' position. Rather unexpectedly, the Leu202-->Phe and Leu202-->Tyr mutations, which were expected to decrease the size of the S-1' subsite, resulted in a large increase in activity towards dipeptide substrates with Phe in the P-1' position. This is probably due to the fact that 202Phe and 202Tyr adopt a second possible rotamer that opens up the subsite compared to Leu202, and also favours interactions with the substrate. To validate these results, we constructed variants of Thermolysin with changes in the S-1' subsite. Thermolysin and TLP-ste variants with identical S-1' subsites were highly similar in terms of their preference for Phe vs. Leu in the P-1' position.
-
structural determinants of the stability of Thermolysin like proteinases
Nature Structural & Molecular Biology, 1995Co-Authors: V G H Eijsink, O R Veltman, W Aukema, G Vriend, G VenemaAbstract:Thermolysin is a member of a family of homologous proteinases which differ in their resistance to thermally induced unfolding and subsequent autolytic degradation. Site-directed mutagenesis studies of the Thermolysin-like proteinase (TLP) from Bacillus stearothermophilus (TLP-ste) show that its reduced resistance to thermally induced autolysis, as compared to Thermolysin, is due to only some of the 44 naturally occurring amino-acid differences between them. In fact TLP-ste becomes more resistant than Thermolysin by mutation of just a few of these amino-acids. The crucial differences are all localized to a solvent-exposed region in the N-terminal domain of TLP-ste.
-
the essential dynamics of Thermolysin confirmation of the hinge bending motion and comparison of simulations in vacuum and water
Proteins, 1995Co-Authors: D M F Van Aalten, V G H Eijsink, G Vriend, Andrea Amadei, Antonius B M Linssen, Herman J C BerendsenAbstract:Comparisons of the crystal structures of Thermolysin and the Thermolysin-like protease produced by B. cereus have recently led to the hypothesis that neutral proteases undergo a hinge-bending motion. We have investigated this hypothesis by analyzing molecular dynamics simulations of Thermolysin in vacuum and water, using the essential dynamics method. This method is able to extract large concerted atomic motions of biological importance from a molecular dynamics trajectory. The analysis of the Thermolysin trajectories indeed revealed a large rigid body hinge-bending motion of the N-terminal and C-terminal domains, similar to the motion hypothesized from the crystal structure comparisons. In addition, it appeared that the essential dynamics properties derived from the vacuum simulation were similar to those derived from the solvent simulation. (C) 1995 Wiley-Liss, Inc.
