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			22247451 962

1astA	Bound:_ZN	Unbound	Unbound		Unbound	Unbound	Unbound
1iaaA	Analogue:_CU	Unbound	Unbound		Unbound	Unbound	Unbound
1iabA	Analogue:_CO	Unbound	Unbound		Unbound	Unbound	Unbound
1iacA	Analogue:_HG	Unbound	Unbound		Unbound	Unbound	Unbound
1iadA	Unbound	Unbound	Unbound		Unbound	Unbound	Unbound
1iaeA	Analogue:_NI	Unbound	Unbound		Unbound	Unbound	Unbound
1qjiA	Bound:_ZN	Unbound	Unbound		Unbound	Unbound	Transition-state-analogue:PKF
1qjjA	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	GLU 93;TYR 149	HIS 92;HIS 96;HIS 102;TYR 149(Zinc binding)
1iaaA	GLU 93;TYR 149	HIS 92;HIS 96;HIS 102;TYR 149(Zinc binding)
1iabA	GLU 93;TYR 149	HIS 92;HIS 96;HIS 102;TYR 149(Zinc binding)
1iacA	GLU 93;TYR 149	HIS 92;HIS 96;HIS 102;TYR 149(Zinc binding)
1iadA	GLU 93;TYR 149	HIS 92;HIS 96;HIS 102;TYR 149(Zinc binding)
1iaeA	GLU 93;TYR 149	HIS 92;HIS 96;HIS 102;TYR 149(Zinc binding)
1qjiA	GLU 93;TYR 149	HIS 92;HIS 96;HIS 102;TYR 149(Zinc binding)
1qjjA	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.

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