DB code: S00175

RLCP classification	1.15.60000.85 : Hydrolysis
CATH domain	3.10.450.30 : Nuclear Transport Factor 2; Chain	Catalytic domain
E.C.	3.1.27.10
CSA	1de3
M-CSA	1de3
MACiE

CATH domain	Related DB codes (homologues)
3.10.450.30 : Nuclear Transport Factor 2; Chain	S00174

Uniprot Enzyme Name
UniprotKB	Protein name	Synonyms	Pfam
P00655	Ribonuclease alpha-sarcin	EC 3.1.27.10 rRNA endonuclease	PF00545 (Ribonuclease) [Graphical View]

KEGG enzyme name
rRNA endonuclease alpha-sarcin

UniprotKB: Accession Number	Entry name	Activity	Subunit	Subcellular location	Cofactor
P00655	RNAS_ASPGI	Hydrolysis of the phosphodiester linkage between guanosine and adenosine residues at one specific position in 28S rRNA from rat ribosomes.		Secreted.

KEGG Pathways
Map code	Pathways	E.C.

Compound table
	Substrates		Products	Intermediates
KEGG-id	C00240	C00001	C02867
E.C.
Compound	rRNA	H2O	Oligoribonucleotide
Type	nucleic acids	H2O	nucleic acids
ChEBI		15377 15377
PubChem		22247451 962 22247451 962

1de3A	Unbound	Unbound	Unbound
1r4yA	Unbound	Unbound	Unbound

Reference for Active-site residues
resource	references	E.C.
literature [1], [2]

Active-site residues
PDB	Catalytic residues	Cofactor-binding residues	Modified residues	Main-chain involved in catalysis	Comment

1de3A	HIS 50;GLU 96;ARG 121;HIS 137
1r4yA	HIS 36;GLU 82;ARG 107;HIS 123

References for Catalytic Mechanism
References	Sections	No. of steps in catalysis
[1]	Fig.7, p.15874	3

References
[1]
Resource
Comments
Medline ID
PubMed ID	9843392
Journal	Biochemistry
Year	1998
Volume	37
Pages	15865-76
Authors	Perez-Canadillas JM, Campos-Olivas R, Lacadena J, Martinez del Pozo A, Gavilanes JG, Santoro J, Rico M, Bruix M
Title	Characterization of pKa values and titration shifts in the cytotoxic ribonuclease alpha-sarcin by NMR. Relationship between electrostatic interactions, structure, and catalytic function.
Related PDB
Related UniProtKB
[2]
Resource
Comments	X-ray crystallography
Medline ID
PubMed ID	10843858
Journal	J Mol Biol
Year	2000
Volume	299
Pages	1061-73
Authors	Perez-Canadillas JM, Santoro J, Campos-Olivas R, Lacadena J, Martinez del Pozo A, Gavilanes JG, Rico M, Bruix M
Title	The highly refined solution structure of the cytotoxic ribonuclease alpha-sarcin reveals the structural requirements for substrate recognition and ribonucleolytic activity.
Related PDB	1de3
Related UniProtKB
[3]
Resource
Comments	catalysis
Medline ID
PubMed ID	11025546
Journal	Proteins
Year	2000
Volume	41
Pages	350-61
Authors	de Antonio C, Martinez del Pozo A, Mancheno JM, Onaderra M, Lacadena J, Martinez-Ruiz A, Perez-Canadillas JM, Bruix M, Gavilanes JG
Title	Assignment of the contribution of the tryptophan residues to the spectroscopic and functional properties of the ribotoxin alpha-sarcin.
Related PDB
Related UniProtKB
[4]
Resource
Comments	catalysis
Medline ID
PubMed ID	11313342
Journal	J Biol Chem
Year	2001
Volume	276
Pages	24075-81
Authors	Rajamohan F, Mao C, Uckun FM
Title	Binding interactions between the active center cleft of recombinant pokeweed antiviral protein and the alpha-sarcin/ricin stem loop of ribosomal RNA.
Related PDB
Related UniProtKB
[5]
Resource
Comments
Medline ID
PubMed ID	12527386
Journal	FEBS Lett
Year	2003
Volume	534
Pages	197-201
Authors	Perez-Canadilllas JM, Garcia-Mayoral MF, Laurents DV, Martinez del Pozo A, Gavilanes JG, Rico M, Bruix M
Title	Tautomeric state of alpha-sarcin histidines. Ndelta tautomers are a common feature in the active site of extracellular microbial ribonucleases.
Related PDB
Related UniProtKB
[6]
Resource
Comments
Medline ID
PubMed ID	14609322
Journal	Biochemistry
Year	2003
Volume	42
Pages	13122-33
Authors	Garcia-Mayoral MF, Perez-Canadillas JM, Santoro J, Ibarra-Molero B, Sanchez-Ruiz JM, Lacadena J, Martinez del Pozo A, Gavilanes JG, Rico M, Bruix M
Title	Dissecting structural and electrostatic interactions of charged groups in alpha-sarcin. An NMR study of some mutants involving the catalytic residues.
Related PDB
Related UniProtKB
[7]
Resource
Comments
Medline ID
PubMed ID	15044731
Journal	Protein Sci
Year	2004
Volume	13
Pages	1000-11
Authors	Garcia-Mayoral MF, Garcia-Ortega L, Lillo MP, Santoro J, Martinez del Pozo A, Gavilanes JG, Rico M, Bruix M
Title	NMR structure of the noncytotoxic alpha-sarcin mutant Delta(7-22): the importance of the native conformation of peripheral loops for activity.
Related PDB	1r4y
Related UniProtKB

Comments
Glu96 and His137 (of 1de3) acts as a general base and general acid, respectively [1]. The transphosphorylation and hydrolysis steps of the reaction follow a concerted in-line mechanism, implying residues acting as a base and an acid located on either side of the scissile bond. Structurally, Glu96 and His137 are on opposite sides of the active center and have the correct geometry for the in-line cleavage of the dinucleotide. According to the literature [1] & [6], the reaction proceeds as follows: (1) Glu96 exists predominantly in the ionized state and acts as a general base abstracting a proton from the 2'-OH group in the ribose ring of the nucleotide. (2) The activated 2'-hydroxy oxygen makes a nucleophilic attack on the scissile phosphorus atom. (3) Arg121 is involved in catalysis, probably as a stabilizer for the negative charge on the phosphoryl group. (4) Meanwhile, His137 remains protonated to serve as general acid and acts as a proton donor to the leaving O5' ester oxygen. (5) 2',3'-cyclic intermediate is formed. (6) Glu96 and His137 act as a general acid and base, respectively. Glu96 activates a water molecule, which attacks on the cyclic intermediate, whereas His137 protonates the leaving 2'-oxygen.

Comments

Glu96 and His137 (of 1de3) acts as a general base and general acid, respectively [1]. The transphosphorylation and hydrolysis steps of the reaction follow a concerted in-line mechanism, implying residues acting as a base and an acid located on either side of the scissile bond. Structurally, Glu96 and His137 are on opposite sides of the active center and have the correct geometry for the in-line cleavage of the dinucleotide.
According to the literature [1] & [6], the reaction proceeds as follows:
(1) Glu96 exists predominantly in the ionized state and acts as a general base abstracting a proton from the 2'-OH group in the ribose ring of the nucleotide.
(2) The activated 2'-hydroxy oxygen makes a nucleophilic attack on the scissile phosphorus atom.
(3) Arg121 is involved in catalysis, probably as a stabilizer for the negative charge on the phosphoryl group.
(4) Meanwhile, His137 remains protonated to serve as general acid and acts as a proton donor to the leaving O5' ester oxygen.
(5) 2',3'-cyclic intermediate is formed.
(6) Glu96 and His137 act as a general acid and base, respectively. Glu96 activates a water molecule, which attacks on the cyclic intermediate, whereas His137 protonates the leaving 2'-oxygen.

Created	Updated
2002-07-01	2011-11-01