PDBsum entry 1sca

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protein metals links
Serine protease PDB id
Protein chain
274 a.a. *
_CA ×2
Waters ×126
* Residue conservation analysis
PDB id:
Name: Serine protease
Title: Enzyme crystal structure in a neat organic solvent
Structure: Subtilisin carlsberg. Chain: a. Engineered: yes
Source: Bacillus licheniformis. Organism_taxid: 1402
2.00Å     R-factor:   0.156    
Authors: P.A.Fitzpatrick,A.C.U.Steinmetz,D.Ringe,A.M.Klibanov
Key ref: P.A.Fitzpatrick et al. (1993). Enzyme crystal structure in a neat organic solvent. Proc Natl Acad Sci U S A, 90, 8653-8657. PubMed id: 8378343 DOI: 10.1073/pnas.90.18.8653
19-Jul-93     Release date:   31-Jan-94    
Go to PROCHECK summary

Protein chain
Pfam   ArchSchema ?
P00780  (SUBT_BACLI) -  Subtilisin Carlsberg
379 a.a.
274 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 5 residue positions (black crosses)

 Enzyme reactions 
   Enzyme class: E.C.  - Subtilisin.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: Hydrolysis of proteins with broad specificity for peptide bonds, and a preference for a large uncharged residue in P1. Hydrolyzes peptide amides.
 Gene Ontology (GO) functional annotation 
  GO annot!
  Biological process     proteolysis   1 term 
  Biochemical function     serine-type endopeptidase activity     1 term  


DOI no: 10.1073/pnas.90.18.8653 Proc Natl Acad Sci U S A 90:8653-8657 (1993)
PubMed id: 8378343  
Enzyme crystal structure in a neat organic solvent.
P.A.Fitzpatrick, A.C.Steinmetz, D.Ringe, A.M.Klibanov.
The crystal structure of the serine protease subtilisin Carlsberg in anhydrous acetonitrile was determined at 2.3 A resolution. It was found to be essentially identical to the three-dimensional structure of the enzyme in water; the differences observed were smaller than those between two independently determined structures in aqueous solution. The hydrogen bond system of the catalytic triad is intact in acetonitrile. The majority (99 of 119) of enzyme-bound, structural water molecules have such a great affinity to subtilisin that they are not displaced even in anhydrous acetonitrile. Of the 12 enzyme-bound acetonitrile molecules, 4 displace water molecules and 8 bind where no water had been observed before. One-third of all subtilisin-bound acetonitrile molecules reside in the active center, occupying the same region (P1, P2, and P3 binding sites) as the specific protein inhibitor eglin c.

Literature references that cite this PDB file's key reference

  PubMed id Reference
21136535 D.van der Spoel, E.G.Marklund, D.S.Larsson, and C.Caleman (2011).
Proteins, lipids, and water in the gas phase.
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Fragment-based cocktail crystallography by the medical structural genomics of pathogenic protozoa consortium.
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17894371 J.E.Stone, J.C.Phillips, P.L.Freddolino, D.J.Hardy, L.G.Trabuco, and K.Schulten (2007).
Accelerating molecular modeling applications with graphics processors.
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17630120 K.Saeki, K.Ozaki, T.Kobayashi, and S.Ito (2007).
Detergent alkaline proteases: enzymatic properties, genes, and crystal structures.
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17419728 N.M.Micaêlo, and C.M.Soares (2007).
Modeling hydration mechanisms of enzymes in nonpolar and polar organic solvents.
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18025587 P.Arriagada-Strodthoff, S.Karboune, R.J.Neufeld, and S.Kermasha (2007).
Optimization of chlorophyllase-catalyzed hydrolysis of chlorophyll in monophasic organic solvent media.
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17461426 Y.Wine, N.Cohen-Hadar, A.Freeman, and F.Frolow (2007).
Elucidation of the mechanism and end products of glutaraldehyde crosslinking reaction by X-ray structure analysis.
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PDB codes: 2htx 2hu1
17187678 B.Castillo, V.Bansal, A.Ganesan, P.Halling, F.Secundo, A.Ferrer, K.Griebenow, and G.Barletta (2006).
On the activity loss of hydrolases in organic solvents: II. a mechanistic study of subtilisin Carlsberg.
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16329143 T.Matsubara, R.Fujita, S.Sugiyama, and K.Kawashiro (2006).
Stability of protease in organic solvent: structural identification by solid-state NMR of lyophilized papain before and after 1-propanol treatment and the corresponding enzymatic activities.
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15272162 C.H.Wang, M.K.Balasubramanian, and T.Dokland (2004).
Structure, crystal packing and molecular dynamics of the calponin-homology domain of Schizosaccharomyces pombe Rng2.
  Acta Crystallogr D Biol Crystallogr, 60, 1396-1403.
PDB codes: 1p2x 1p5s
15557256 G.Fuentes, A.Ballesteros, and C.S.Verma (2004).
Specificity in lipases: a computational study of transesterification of sucrose.
  Protein Sci, 13, 3092-3103.  
15298890 L.Yang, J.S.Dordick, and S.Garde (2004).
Hydration of enzyme in nonaqueous media is consistent with solvent dependence of its activity.
  Biophys J, 87, 812-821.  
12609866 C.M.Soares, V.H.Teixeira, and A.M.Baptista (2003).
Protein structure and dynamics in nonaqueous solvents: insights from molecular dynamics simulation studies.
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12829506 L.He, S.André, H.C.Siebert, H.Helmholz, B.Niemeyer, and H.J.Gabius (2003).
Detection of ligand- and solvent-induced shape alterations of cell-growth-regulatory human lectin galectin-1 in solution by small angle neutron and x-ray scattering.
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11835510 D.Vitkup, D.Ringe, M.Karplus, and G.A.Petsko (2002).
Why protein R-factors are so large: a self-consistent analysis.
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11857281 I.Montañez-Clemente, E.Alvira, M.Macias, A.Ferrer, M.Fonceca, J.Rodriguez, A.Gonzalez, and G.Barletta (2002).
Enzyme activation in organic solvents: co-lyophilization of subtilisin Carlsberg with methyl-beta-cyclodextrin renders an enzyme catalyst more active than the cross-linked enzyme crystals.
  Biotechnol Bioeng, 78, 53-59.  
11863433 X.Siebert, and G.Hummer (2002).
Hydrophobicity maps of the N-peptide coiled coil of HIV-1 gp41.
  Biochemistry, 41, 2956-2961.  
11342057 V.V.Gorbatchuk, M.A.Ziganshin, N.A.Mironov, and B.N.Solomonov (2001).
Homotropic cooperative binding of organic solvent vapors by solid trypsin.
  Biochim Biophys Acta, 1545, 326-338.  
11114170 A.D.Miranker (2000).
Mass spectrometry of proteins of known mass.
  Proc Natl Acad Sci U S A, 97, 14025-14027.  
10636235 D.B.Jordan, and G.S.Basarab (2000).
Binding dynamics of two water molecules constrained within the scytalone dehydratase binding pocket.
  Bioorg Med Chem Lett, 10, 23-26.  
10679381 G.A.Petsko, and D.Ringe (2000).
Observation of unstable species in enzyme-catalyzed transformations using protein crystallography.
  Curr Opin Chem Biol, 4, 89-94.  
10898867 J.S.Shin, S.Luque, and A.M.Klibanov (2000).
Improving lipase enantioselectivity in organic solvents by forming substrate salts with chiral agents.
  Biotechnol Bioeng, 69, 577-583.  
10651278 A.C.English, S.H.Done, L.S.Caves, C.R.Groom, and R.E.Hubbard (1999).
Locating interaction sites on proteins: the crystal structure of thermolysin soaked in 2% to 100% isopropanol.
  Proteins, 37, 628-640.
PDB codes: 1tli 1tlx 2tli 2tlx 3tli 4tli 5tli 6tli 7tli 8tli
10021400 D.Häring, and P.Schreier (1999).
Cross-linked enzyme crystals.
  Curr Opin Chem Biol, 3, 35-38.  
10398928 D.Ringe, and C.Mattos (1999).
Analysis of the binding surfaces of proteins.
  Med Res Rev, 19, 321-331.  
10226504 D.S.Dwyer (1999).
Molecular simulation of the effects of alcohols on peptide structure.
  Biopolymers, 49, 635-645.  
9990001 E.Zacharis, P.J.Halling, and D.G.Rees (1999).
Volatile buffers can override the "pH memory" of subtilisin catalysis in organic media.
  Proc Natl Acad Sci U S A, 96, 1201-1205.  
10665832 G.K.Farber (1999).
New approaches to rational drug design.
  Pharmacol Ther, 84, 327-332.  
10404234 G.Vecchio, F.Zambianchi, P.Zacchetti, F.Secundo, and G.Carrea (1999).
Fourier-transform infrared spectroscopy study of dehydrated lipases from candida antarctica B and pseudomonas cepacia
  Biotechnol Bioeng, 64, 545-551.  
10099631 P.Pepin, and R.Lortie (1999).
Influence of water activity on the enantioselective esterification of (R,S)-ibuprofen by Candida antarctica lipase B in solventless media.
  Biotechnol Bioeng, 63, 502-505.  
10468562 X.G.Gao, E.Maldonado, R.Pérez-Montfort, G.Garza-Ramos, Gómez-Puyou, A.Gómez-Puyou, and A.Rodríguez-Romero (1999).
Crystal structure of triosephosphate isomerase from Trypanosoma cruzi in hexane.
  Proc Natl Acad Sci U S A, 96, 10062-10067.
PDB code: 1ci1
9449362 B.Yu, and D.L.Caspar (1998).
Structure of cubic insulin crystals in glucose solutions.
  Biophys J, 74, 616-622.  
9789015 J.L.Schmitke, L.J.Stern, and A.M.Klibanov (1998).
Comparison of x-ray crystal structures of an acyl-enzyme intermediate of subtilisin Carlsberg formed in anhydrous acetonitrile and in water.
  Proc Natl Acad Sci U S A, 95, 12918-12923.
PDB codes: 1be6 1be8
18636650 C.R.Wescott, and A.M.Klibanov (1997).
Thermodynamic analysis of solvent effect on substrate specificity of lyophilized enzymes suspended in organic media.
  Biotechnol Bioeng, 56, 340-344.  
9113975 J.L.Schmitke, L.J.Stern, and A.M.Klibanov (1997).
The crystal structure of subtilisin Carlsberg in anhydrous dioxane and its comparison with those in water and acetonitrile.
  Proc Natl Acad Sci U S A, 94, 4250-4255.
PDB code: 1af4
18634023 K.Griebenow, and A.M.Klibanov (1997).
Can conformational changes be responsible for solvent and excipient effects on the catalytic behavior of subtilisin Carlsberg in organic solvents?
  Biotechnol Bioeng, 53, 351-362.  
9341205 S.L.Lee, R.S.Alexander, A.Smallwood, R.Trievel, L.Mersinger, P.C.Weber, and C.Kettner (1997).
New inhibitors of thrombin and other trypsin-like proteases: hydrogen bonding of an aromatic cyano group with a backbone amide of the P1 binding site replaces binding of a basic side chain.
  Biochemistry, 36, 13180-13186.
PDB code: 1auj
8973167 A.A.Fedorov, D.Joseph-McCarthy, E.Fedorov, D.Sirakova, I.Graf, and S.C.Almo (1996).
Ionic interactions in crystalline bovine pancreatic ribonuclease A.
  Biochemistry, 35, 15962-15979.
PDB codes: 1rno 1rnq 1rnw 1rnx 1rny 1rnz
8913568 G.H.Peters, D.M.van Aalten, O.Edholm, S.Toxvaerd, and R.Bywater (1996).
Dynamics of proteins in different solvent systems: analysis of essential motion in lipases.
  Biophys J, 71, 2245-2255.  
18627092 H.Noritomi, O.Almarsson, G.L.Barletta, and A.M.Klibanov (1996).
The influence of the mode of enzyme preparation on enzymatic enantioselectivity in organic solvents and its temperature dependence.
  Biotechnol Bioeng, 51, 95-99.  
8924626 M.J.Bodkin, and J.M.Goodfellow (1996).
Hydrophobic solvation in aqueous trifluoroethanol solution.
  Biopolymers, 39, 43-50.  
18623557 O.Almarsson, and A.M.Klibanov (1996).
Remarkable activation of enzymes in nonaqueous media by denaturing organic cosolvents.
  Biotechnol Bioeng, 49, 87-92.  
21121013 Y.J.Zheng, and R.L.Ornstein (1996).
Molecular dynamics of subtilisin Carlsberg in aqueous and nonaqueous solutions.
  Biopolymers, 38, 791-799.  
8749372 D.Ringe (1995).
What makes a binding site a binding site?
  Curr Opin Struct Biol, 5, 825-829.  
18623476 J.B.van Tol, J.A.Jongejan, J.A.Duine, H.G.Kierkels, E.F.Geladé, F.Mosterd, W.J.van der Tweel, and J.Kamphuis (1995).
Thermodynamic and kinetic parameters of lipase-catalyzed ester hydrolysis in biphasic systems with varying organic solvents.
  Biotechnol Bioeng, 48, 179-189.  
18623368 J.B.van Tol, R.M.Stevens, W.J.Veldhuizen, J.A.Jongejan, and J.A.Duine (1995).
Do organic solvents affect the catalytic properties of lipase? Intrinsic kinetic parameters of lipases in ester hydrolysis and formation in various organic solvents.
  Biotechnol Bioeng, 47, 71-81.  
18623235 J.O.Rich, B.A.Bedell, and J.S.Dordick (1995).
Controlling enzyme-catalyzed regioselectivity in sugar ester synthesis.
  Biotechnol Bioeng, 45, 426-434.  
7607252 P.Polverino de Laureto, V.De Filippis, E.Scaramella, M.Zambonin, and A.Fontana (1995).
Limited proteolysis of lysozyme in trifluoroethanol. Isolation and characterization of a partially active enzyme derivative.
  Eur J Biochem, 230, 779-787.  
7662108 S.T.Chen, S.Y.Chen, C.C.Tu, S.H.Chiou, and K.T.Wang (1995).
Physicochemical properties of alkaline serine proteases in alcohol.
  J Protein Chem, 14, 205-215.  
7937734 M.B.Eisen, D.C.Wiley, M.Karplus, and R.E.Hubbard (1994).
HOOK: a program for finding novel molecular architectures that satisfy the chemical and steric requirements of a macromolecule binding site.
  Proteins, 19, 199-221.  
7948673 M.Norin, F.Haeffner, K.Hult, and O.Edholm (1994).
Molecular dynamics simulations of an enzyme surrounded by vacuum, water, or a hydrophobic solvent.
  Biophys J, 67, 548-559.  
7764428 R.Peters, and R.C.McKinstry (1994).
Three-dimensional modeling and drug development. Has "rational" drug design arrived?
  Biotechnology (N Y), 12, 147-150.  
The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB codes are shown on the right.