DB code: S00394

RLCP classification 1.13.11100.261 : Hydrolysis
CATH domain 3.40.390.10 : Collagenase (Catalytic Domain) Catalytic domain
E.C. 3.4.24.21
CSA 1ast
M-CSA 1ast
MACiE

CATH domain Related DB codes (homologues)
3.40.390.10 : Collagenase (Catalytic Domain) S00395 S00397 S00398 S00399 D00232 D00236 M00101

Uniprot Enzyme Name
UniprotKB Protein name Synonyms MEROPS Pfam
P07584 Astacin
EC 3.4.24.21
Crayfish small molecule proteinase
M12.001 (Metallo)
PF01400 (Astacin)
[Graphical View]

KEGG enzyme name
astacin
Astacus proteinase
crayfish small-molecule proteinase

UniprotKB: Accession Number Entry name Activity Subunit Subcellular location Cofactor
P07584 ASTA_ASTFL Hydrolysis of peptide bonds in substrates containing five or more amino acids, preferentially with Ala in P1'', and Pro in P2''. Monomer. Binds 1 zinc ion per subunit.

KEGG Pathways
Map code Pathways E.C.

Compound table
Cofactors Substrates Products Intermediates
KEGG-id C00038 C00017 C00012 C00001 C00017 C00012
E.C.
Compound Zinc Protein Peptide H2O Protein Peptide
Type heavy metal peptide/protein peptide/protein H2O peptide/protein peptide/protein
ChEBI 29105
15377
PubChem 32051
962
22247451
1astA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Bound:_ZN Unbound Unbound Unbound Unbound Unbound
1iaaA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Analogue:_CU Unbound Unbound Unbound Unbound Unbound
1iabA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Analogue:_CO Unbound Unbound Unbound Unbound Unbound
1iacA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Analogue:_HG Unbound Unbound Unbound Unbound Unbound
1iadA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Unbound Unbound Unbound Unbound Unbound
1iaeA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Analogue:_NI Unbound Unbound Unbound Unbound Unbound
1qjiA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Bound:_ZN Unbound Unbound Unbound Unbound Transition-state-analogue:PKF
1qjjA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Bound:_ZN Unbound Unbound Unbound Analogue:PRO-LEU-GLY-HOA Unbound

Reference for Active-site residues
resource references E.C.
Swiss-prot;P07584

Active-site residues
PDB Catalytic residues Cofactor-binding residues Modified residues Main-chain involved in catalysis Comment
1astA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain GLU 93;TYR 149 HIS 92;HIS 96;HIS 102;TYR 149(Zinc binding)
1iaaA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain GLU 93;TYR 149 HIS 92;HIS 96;HIS 102;TYR 149(Zinc binding)
1iabA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain GLU 93;TYR 149 HIS 92;HIS 96;HIS 102;TYR 149(Zinc binding)
1iacA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain GLU 93;TYR 149 HIS 92;HIS 96;HIS 102;TYR 149(Zinc binding)
1iadA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain GLU 93;TYR 149 HIS 92;HIS 96;HIS 102;TYR 149(Zinc binding)
1iaeA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain GLU 93;TYR 149 HIS 92;HIS 96;HIS 102;TYR 149(Zinc binding)
1qjiA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain GLU 93;TYR 149 HIS 92;HIS 96;HIS 102;TYR 149(Zinc binding)
1qjjA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain GLU 93;TYR 149 HIS 92;HIS 96;HIS 102;TYR 149(Zinc binding)

References for Catalytic Mechanism
References Sections No. of steps in catalysis
[6]
p.17113-17117
[7]
p.321-325
[9]
Fig.3, p.673-674 3
[10]
Scheme 3, p.4955-4958
[11]
p.227

References
[1]
Resource
Comments X-ray crystallography
Medline ID
PubMed ID 1319561
Journal Nature
Year 1992
Volume 358
Pages 164-7
Authors Bode W, Gomis-Ruth FX, Huber R, Zwilling R, Stocker W
Title Structure of astacin and implications for activation of astacins and zinc-ligation of collagenases.
Related PDB 1ast
Related UniProtKB P07584
[2]
Resource
Comments X-ray crystallography (1.8 Angstroms)
Medline ID
PubMed ID 8445658
Journal J Mol Biol
Year 1993
Volume 229
Pages 945-68
Authors Gomis-Ruth FX, Stocker W, Huber R, Zwilling R, Bode W
Title Refined 1.8 A X-ray crystal structure of astacin, a zinc-endopeptidase from the crayfish Astacus astacus L. Structure determination, refinement, molecular structure and comparison with thermolysin.
Related PDB 1iac 1iad
Related UniProtKB P07584
[3]
Resource
Comments
Medline ID
PubMed ID 8248170
Journal Proc Natl Acad Sci U S A
Year 1993
Volume 90
Pages 10783-7
Authors Wolfsberg TG, Bazan JF, Blobel CP, Myles DG, Primakoff P, White JM
Title The precursor region of a protein active in sperm-egg fusion contains a metalloprotease and a disintegrin domain: structural, functional, and evolutionary implications.
Related PDB
Related UniProtKB
[4]
Resource
Comments
Medline ID
PubMed ID 8262184
Journal FEBS Lett
Year 1993
Volume 335
Pages 361-6
Authors Corbeil D, Milhiet PE, Simon V, Ingram J, Kenny AJ, Boileau G, Crine P
Title Rat endopeptidase-24.18 alpha subunit is secreted into the culture medium as a zymogen when expressed by COS-1 cells.
Related PDB
Related UniProtKB
[5]
Resource
Comments
Medline ID
PubMed ID 8198548
Journal Biochem J
Year 1994
Volume 300
Pages 37-43
Authors Milhiet PE, Corbeil D, Simon V, Kenny AJ, Crine P, Boileau G
Title Expression of rat endopeptidase-24.18 in COS-1 cells: membrane topology and activity.
Related PDB
Related UniProtKB
[6]
Resource
Comments X-ray crystallography
Medline ID
PubMed ID 8006015
Journal J Biol Chem
Year 1994
Volume 269
Pages 17111-7
Authors Gomis-Ruth FX, Grams F, Yiallouros I, Nar H, Kusthardt U, Zwilling R, Bode W, Stocker W
Title Crystal structures, spectroscopic features, and catalytic properties of cobalt(II), copper(II), nickel(II), and mercury(II) derivatives of the zinc endopeptidase astacin. A correlation of structure and proteolytic activity.
Related PDB 1iaa 1iab 1iae
Related UniProtKB
[7]
Resource
Comments
Medline ID
PubMed ID 7674929
Journal Methods Enzymol
Year 1995
Volume 248
Pages 305-25
Authors Stocker W, Zwilling R
Title Astacin.
Related PDB
Related UniProtKB
[8]
Resource
Comments
Medline ID
PubMed ID 8740360
Journal Structure
Year 1996
Volume 4
Pages 375-86
Authors Dhanaraj V, Ye QZ, Johnson LL, Hupe DJ, Ortwine DF, Dunbar JB Jr, Rubin JR, Pavlovsky A, Humblet C, Blundell TL
Title X-ray structure of a hydroxamate inhibitor complex of stromelysin catalytic domain and its comparison with members of the zinc metalloproteinase superfamily.
Related PDB
Related UniProtKB
[9]
Resource
Comments
Medline ID
PubMed ID 8756323
Journal Nat Struct Biol
Year 1996
Volume 3
Pages 671-5
Authors Grams F, Dive V, Yiotakis A, Yiallouros I, Vassiliou S, Zwilling R, Bode W, Stocker W
Title Structure of astacin with a transition-state analogue inhibitor.
Related PDB 1qji 1qjj
Related UniProtKB
[10]
Resource
Comments
Medline ID
PubMed ID 9125517
Journal Biochemistry
Year 1997
Volume 36
Pages 4949-58
Authors Mock WL, Yao J
Title Kinetic characterization of the serralysins: a divergent catalytic mechanism pertaining to astacin-type metalloproteases.
Related PDB
Related UniProtKB
[11]
Resource
Comments
Medline ID
PubMed ID 11078883
Journal FEBS Lett
Year 2000
Volume 484
Pages 224-8
Authors Yiallouros I, Grosse Berkhoff E, Stocker W
Title The roles of Glu93 and Tyr149 in astacin-like zinc peptidases.
Related PDB
Related UniProtKB
[12]
Resource
Comments
Medline ID
PubMed ID 12441103
Journal J Mol Biol
Year 2002
Volume 324
Pages 237-46
Authors Yiallouros I, Kappelhoff R, Schilling O, Wegmann F, Helms MW, Auge A, Brachtendorf G, Berkhoff EG, Beermann B, Hinz HJ, Konig S, Peter-Katalinic J, Stocker W
Title Activation mechanism of pro-astacin: role of the pro-peptide, tryptic and autoproteolytic cleavage and importance of precise amino-terminal processing.
Related PDB
Related UniProtKB

Comments
This enzyme belongs to the peptidase family-M12A.
Although an alternative machanism has been reported [10], in which Tyr149, instead of Glu93, functions as a general base to deprotonate a solvent water molecule, other experimental study such as X-ray crystallography and mutagenesis ([9] & [11]) confirmed that Glu93 and Tyr149 act as a general base and a stabilizer of the transition state, respectively.
In the free enzyme, the catalytic zinc is liganded by three imidazol nitrogen atoms from the histidines (His92, His96 & His102) and a oxygen atom of solvent water molecule, and another oxygen atom from the sidechain of Tyr149, forming a trigonal-bipyramidal coordination sphere (see [2], [6] & [7]). Upon substrate binding, some rearrangement of the Tyr149 residue seems to occur, suggesting this is a 'tyrosine switch', according to the literature [9].
Thus, the catalytic mechanism proceeds as follows (see [9], [11]):
(1) Glu93 activates the catalytic water, which in turn makes a nucleophilic attack on the carbonyl carbon atom.
(2) Tyr149 acts as an electrophile, to bind and to stabilize the tetrahedral carboxyanion of the transition state.
(3) The protonated carboxylate of Glu93 then protonates the leaving amide group.

Created Updated
2002-09-27 2009-02-26