PDBsum entry 4tmn

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Hydrolase/hydrolase inhibitor PDB id
Protein chain
316 a.a. *
_CA ×4
Waters ×162
* Residue conservation analysis
PDB id:
Name: Hydrolase/hydrolase inhibitor
Title: Slow-and fast-binding inhibitors of thermolysin display diff modes of binding. Crystallographic analysis of extended phosphonamidate transition-state analogues
Structure: Thermolysin. Chain: e. Engineered: yes
Source: Bacillus thermoproteolyticus. Organism_taxid: 1427
Biol. unit: Tetramer (from PQS)
1.70Å     R-factor:   0.170    
Authors: H.M.Holden,D.E.Tronrud,A.F.Monzingo,L.H.Weaver,B.W.Matthews
Key ref:
H.M.Holden et al. (1987). Slow- and fast-binding inhibitors of thermolysin display different modes of binding: crystallographic analysis of extended phosphonamidate transition-state analogues. Biochemistry, 26, 8542-8553. PubMed id: 3442675 DOI: 10.1021/bi00400a008
29-Jun-87     Release date:   09-Jan-89    
Go to PROCHECK summary

Protein chain
Pfam   ArchSchema ?
P00800  (THER_BACTH) -  Thermolysin
548 a.a.
316 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 2 residue positions (black crosses)

 Enzyme reactions 
   Enzyme class: E.C.  - Thermolysin.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: Preferential cleavage: Xaa-|-Leu > Xaa-|-Phe.
      Cofactor: Ca(2+); Zn(2+)
 Gene Ontology (GO) functional annotation 
  GO annot!
  Biological process     proteolysis   1 term 
  Biochemical function     metalloendopeptidase activity     1 term  


DOI no: 10.1021/bi00400a008 Biochemistry 26:8542-8553 (1987)
PubMed id: 3442675  
Slow- and fast-binding inhibitors of thermolysin display different modes of binding: crystallographic analysis of extended phosphonamidate transition-state analogues.
H.M.Holden, D.E.Tronrud, A.F.Monzingo, L.H.Weaver, B.W.Matthews.
The modes of binding to thermolysin of two phosphonamidate peptide inhibitors, carbobenzoxy-GlyP-L-Leu-L-Leu (ZGPLL) and carbobenzoxy-L-PheP-L-Leu-L-Ala (ZFPLA), have been determined by X-ray crystallography and refined at high resolution to crystallographic R-values of 17.7% and 17.0%, respectively. (GlyP is used to indicate that the trigonal carbon of the peptide linkage is replaced by the tetrahedral phosphorus of a phosphonamidate group.). These inhibitors were designed to be structural analogues of the presumed catalytic transition state and are potent inhibitors of thermolysin (ZGPLL, Ki = 9.1 nM; ZFPLA, Ki = 0.068 nM) [Bartlett, P. A., & Marlowe, C. K. (1987) Biochemistry (following paper in this issue)]. ZFPLA binds to thermolysin in the manner expected for the transition state and, for the first time, provides direct support for the presumed mode of binding of extended substrates in the S2 subsite. The mode of binding of ZFPLA displays all the interactions that are presumed to stabilize the transition state and supports the postulated mechanism of catalysis [Hangauer, D. G., Monzingo, A. F., & Matthews, B. W. (1984) Biochemistry 23, 5730-5741]. The two oxygens of the phosphonamidate moiety are liganded to the zinc to give overall pentacoordination of the metal. For the second inhibitor the situation is different. Although both ZFPLA and ZGPLL have similar modes of binding in the S1' and S2' subsites, the configurations of the carbobenzoxy-Phe and carbobenzoxy-Gly moieties are different. For ZFPLA the carbonyl group of the carbobenzoxy group is hydrogen bonded directly to the enzyme, whereas in ZGPLL the carbonyl group is rotated 117 degrees, and there is a water molecule interposed between the inhibitor and the enzyme. For ZGPLL only one of the phosphonamidate oxygens is liganded to the zinc. Correlated with the change in inhibitor-zinc ligation from monodentate in ZGPLL to bidentate in ZFPLA there is an increase in the phosphorus-nitrogen bond length of about 0.25 A, strongly suggesting that the phosphonamide nitrogen in ZFPLA is cationic, analogous to the doubly protonated nitrogen of the transition state. The observation that the nitrogen of ZFPLA appears to donate two hydrogen bonds to the protein also indicates that it is cationic. The different configurations adopted by the respective inhibitors are correlated with large differences in their kinetics of binding [Bartlett, P. A., & Marlowe, C. K. (1987) Biochemistry (following paper in this issue)]. These differences in kinetics are not associated with any significant conformational change on the part of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Literature references that cite this PDB file's key reference

  PubMed id Reference
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Structural basis for specific cleavage of Lys 63-linked polyubiquitin chains.
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PDB codes: 2znr 2znv
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Swapping the substrate specificities of the neuropeptidases neurolysin and thimet oligopeptidase.
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PDB codes: 2o36 2o3e
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PDB code: 1e1h
14998993 K.Ray, C.S.Hines, J.Coll-Rodriguez, and D.W.Rodgers (2004).
Crystal structure of human thimet oligopeptidase provides insight into substrate recognition, regulation, and localization.
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PDB code: 1s4b
15592454 M.A.Breidenbach, and A.T.Brunger (2004).
Substrate recognition strategy for botulinum neurotoxin serotype A.
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PDB codes: 1xtf 1xtg
14993599 M.Fujinaga, M.M.Cherney, H.Oyama, K.Oda, and M.N.James (2004).
The molecular structure and catalytic mechanism of a novel carboxyl peptidase from Scytalidium lignicolum.
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PDB codes: 1s2b 1s2k
14754895 P.Towler, B.Staker, S.G.Prasad, S.Menon, J.Tang, T.Parsons, D.Ryan, M.Fisher, D.Williams, N.A.Dales, M.A.Patane, and M.W.Pantoliano (2004).
ACE2 X-ray structures reveal a large hinge-bending motion important for inhibitor binding and catalysis.
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PDB codes: 1r42 1r4l
15027050 S.Swaminathan, S.Eswaramoorthy, and D.Kumaran (2004).
Structure and enzymatic activity of botulinum neurotoxins.
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12832763 M.Selkti, A.Tomas, J.F.Gaucher, T.Prangé, M.C.Fournié-Zaluski, H.Chen, and B.P.Roques (2003).
Interactions of a new alpha-aminophosphinic derivative inside the active site of TLN (thermolysin): a model for zinc-metalloendopeptidase inhibition.
  Acta Crystallogr D Biol Crystallogr, 59, 1200-1205.
PDB codes: 1no0 1os0
11839307 J.W.Arndt, B.Hao, V.Ramakrishnan, T.Cheng, S.I.Chan, and M.K.Chan (2002).
Crystal structure of a novel carboxypeptidase from the hyperthermophilic archaeon Pyrococcus furiosus.
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PDB codes: 1k9x 1ka2 1ka4
12406750 K.Miyamoto, E.Nukui, M.Hirose, F.Nagai, T.Sato, Y.Inamori, and H.Tsujibo (2002).
A metalloprotease (MprIII) involved in the chitinolytic system of a marine bacterium, Alteromonas sp. strain O-7.
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11559368 Kreij, B.van den Burg, O.R.Veltman, G.Vriend, G.Venema, and V.G.Eijsink (2001).
The effect of changing the hydrophobic S1' subsite of thermolysin-like proteases on substrate specificity.
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11248043 C.K.Brown, K.Madauss, W.Lian, M.R.Beck, W.D.Tolbert, and D.W.Rodgers (2001).
Structure of neurolysin reveals a deep channel that limits substrate access.
  Proc Natl Acad Sci U S A, 98, 3127-3132.
PDB code: 1i1i
11223512 T.Hori, T.Kumasaka, M.Yamamoto, N.Nonaka, N.Tanaka, Y.Hashimoto, U.Ueki, and K.Takio (2001).
Structure of a new 'aspzincin' metalloendopeptidase from Grifola frondosa: implications for the catalytic mechanism and substrate specificity based on several different crystal forms.
  Acta Crystallogr D Biol Crystallogr, 57, 361-368.
PDB codes: 1g12 1ge5 1ge6 1ge7
10753902 J.E.Jackman, C.A.Fierke, L.N.Tumey, M.Pirrung, T.Uchiyama, S.H.Tahir, O.Hindsgaul, and C.R.Raetz (2000).
Antibacterial agents that target lipid A biosynthesis in gram-negative bacteria. Inhibition of diverse UDP-3-O-(r-3-hydroxymyristoyl)-n-acetylglucosamine deacetylases by substrate analogs containing zinc binding motifs.
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  10850800 K.M.Holtz, B.Stec, J.K.Myers, S.M.Antonelli, T.S.Widlanski, and E.R.Kantrowitz (2000).
Alternate modes of binding in two crystal structures of alkaline phosphatase-inhibitor complexes.
  Protein Sci, 9, 907-915.
PDB codes: 1ew8 1ew9
10713513 M.Fujinaga, M.M.Cherney, N.I.Tarasova, P.A.Bartlett, J.E.Hanson, and M.N.James (2000).
Structural study of the complex between human pepsin and a phosphorus-containing peptidic -transition-state analog.
  Acta Crystallogr D Biol Crystallogr, 56, 272-279.
PDB code: 1qrp
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
  10217773 C.M.Jung, O.Matsushita, S.Katayama, J.Minami, J.Sakurai, and A.Okabe (1999).
Identification of metal ligands in the Clostridium histolyticum ColH collagenase.
  J Bacteriol, 181, 2816-2822.  
10074467 I.A.Lessard, and C.T.Walsh (1999).
Mutational analysis of active-site residues of the enterococcal D-ala-D-Ala dipeptidase VanX and comparison with Escherichia coli D-ala-D-Ala ligase and D-ala-D-Ala carboxypeptidase VanY.
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10611646 J.D.Tyndall, and D.P.Fairlie (1999).
Conformational homogeneity in molecular recognition by proteolytic enzymes.
  J Mol Recognit, 12, 363-370.  
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Thermolysin and mitochondrial processing peptidase: how far structure-functional convergence goes.
  Protein Sci, 8, 2537-2540.  
10092869 S.Kunugi, Y.Yanagi, and K.Oda (1999).
Studies on the formation and stability of a complex between Streptomyces proteinaceous metalloprotease inhibitor and thermolysin.
  Eur J Biochem, 259, 815-820.  
  10082367 I.L.Alberts, K.Nadassy, and S.J.Wodak (1998).
Analysis of zinc binding sites in protein crystal structures.
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9348662 C.L.Perrin, and J.B.Nielson (1997).
"Strong" hydrogen bonds in chemistry and biology.
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Protein clefts in molecular recognition and function.
  Protein Sci, 5, 2438-2452.  
7561976 C.McMartin, and R.S.Bohacek (1995).
Flexible matching of test ligands to a 3D pharmacophore using a molecular superposition force field: comparison of predicted and experimental conformations of inhibitors of three enzymes.
  J Comput Aided Mol Des, 9, 237-250.  
  8535232 D.R.Holland, A.C.Hausrath, D.Juers, and B.W.Matthews (1995).
Structural analysis of zinc substitutions in the active site of thermolysin.
  Protein Sci, 4, 1955-1965.
PDB codes: 1lna 1lnb 1lnc 1lnd 1lne 1lnf
  7795520 J.Shen, and J.Wendoloski (1995).
Binding of phosphorus-containing inhibitors to thermolysin studied by the Poisson-Boltzmann method.
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7964924 R.L.DesJarlais, and J.S.Dixon (1994).
A shape- and chemistry-based docking method and its use in the design of HIV-1 protease inhibitors.
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8473917 C.M.Ho, and G.R.Marshall (1993).
FOUNDATION: a program to retrieve all possible structures containing a user-defined minimum number of matching query elements from three-dimensional databases.
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8081834 M.Izquierdo-Martin, and R.L.Stein (1993).
Mechanistic studies on the inhibition of stromelysin by a peptide phosphonamidate.
  Bioorg Med Chem, 1, 19-26.  
1730223 S.Mangani, P.Carloni, and P.Orioli (1992).
X-ray diffraction study of the interaction between carboxypeptidase A and (S)-(+)-1-amino-2-phenylethyl phosphonic acid.
  Eur J Biochem, 203, 173-177.  
1633827 W.Stark, R.A.Pauptit, K.S.Wilson, and J.N.Jansonius (1992).
The structure of neutral protease from Bacillus cereus at 0.2-nm resolution.
  Eur J Biochem, 207, 781-791.
PDB code: 1npc
2167850 V.Dive, A.Yiotakis, A.Nicolaou, and F.Toma (1990).
Inhibition of Clostridium histolyticum collagenases by phosphonamide peptide inhibitors.
  Eur J Biochem, 191, 685-693.  
2715793 C.Giessner-Prettre, and O.Jacob (1989).
A theoretical study of Zn++ interacting with models of ligands present at the thermolysin active site.
  J Comput Aided Mol Des, 3, 23-37.  
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.