DB code: S00467

RLCP classification	1.15.60000.82 : Hydrolysis
CATH domain	3.90.730.10 : Ribonuclease Rh; Chain A	Catalytic domain
E.C.	3.1.27.1
CSA	1bol
M-CSA	1bol
MACiE

CATH domain	Related DB codes (homologues)

Uniprot Enzyme Name
UniprotKB	Protein name	Synonyms	Pfam	RefSeq
P08056	Ribonuclease Rh	RNase Rh EC 3.1.27.1	PF00445 (Ribonuclease_T2) [Graphical View]
P23540	Ribonuclease MC	RNase MC EC 3.1.27.1	PF00445 (Ribonuclease_T2) [Graphical View]
P80022	Extracellular ribonuclease LE	RNase LE EC 3.1.27.1	PF00445 (Ribonuclease_T2) [Graphical View]	NP_001234195.1 (Protein) NM_001247266.1 (DNA/RNA sequence)
Q9SSV1		RNase NGR3 (Ribonuclease NGR3)	PF00445 (Ribonuclease_T2) [Graphical View]

KEGG enzyme name
ribonuclease T2 ribonuclease II base-non-specific ribonuclease nonbase-specific RNase RNase (non-base specific) non-base specific ribonuclease nonspecific RNase RNase Ms RNase M RNase II Escherichia coli ribonuclease II ribonucleate nucleotido-2'-transferase (cyclizing) acid ribonuclease RNAase CL Escherichia coli ribonuclease I' ribonuclease PP2 ribonuclease N2 ribonuclease M acid RNase ribonnuclease (non-base specific) ribonuclease (non-base specific) RNase T2 ribonuclease PP3 ribonucleate 3'-oligonucleotide hydrolase RNase II ribonuclease U4

KEGG enzyme name

ribonuclease T2
ribonuclease II
base-non-specific ribonuclease
nonbase-specific RNase
RNase (non-base specific)
non-base specific ribonuclease
nonspecific RNase
RNase Ms
RNase M
RNase II
Escherichia coli ribonuclease II
ribonucleate nucleotido-2'-transferase (cyclizing)
acid ribonuclease
RNAase CL
Escherichia coli ribonuclease I' ribonuclease PP2
ribonuclease N2
ribonuclease M
acid RNase
ribonnuclease (non-base specific)
ribonuclease (non-base specific)
RNase T2
ribonuclease PP3
ribonucleate 3'-oligonucleotide hydrolase
RNase II
ribonuclease U4

UniprotKB: Accession Number	Entry name	Activity	Subcellular location
P08056	RNRH_RHINI	Two-stage endonucleolytic cleavage to nucleoside 3''-phosphates and 3''-phosphooligonucleotides with 2'',3''-cyclic phosphate intermediates.
P23540	RNMC_MOMCH	Two-stage endonucleolytic cleavage to nucleoside 3''-phosphates and 3''-phosphooligonucleotides with 2'',3''-cyclic phosphate intermediates.
P80022	RNLE_SOLLC	Two-stage endonucleolytic cleavage to nucleoside 3''-phosphates and 3''-phosphooligonucleotides with 2'',3''-cyclic phosphate intermediates.	Secreted, extracellular space. Secreted, cell wall.
Q9SSV1	Q9SSV1_NICGU

KEGG Pathways
Map code	Pathways	E.C.

Compound table
	Substrates		Products			Intermediates
KEGG-id	C00046	C00001	C00172	C03419	C01199
E.C.
Compound	RNA	H2O	3'-Phosphooligonucleotide	Nucleoside 3'-phosphate	5'-Hydroxyoligonucleotide	2',3'-cyclic phosphate mononucleotide
Type	nucleic acids	H2O	nucleic acids	nucleotide	nucleic acids
ChEBI		15377 15377
PubChem		22247451 962 22247451 962

1bolA	Unbound		Unbound	Unbound	Unbound	Unbound
1bk7A	Unbound		Unbound	Unbound	Unbound	Unbound
1j1fA	Analogue:5GP		Unbound	Unbound	Unbound	Unbound
1j1gA	Analogue:5GP_295		Unbound	Unbound	Unbound	Unbound
1ucaA	Unbound		Unbound	Unbound	Unbound	Unbound
1uccA	Unbound		Unbound	Unbound	Unbound	Unbound
1ucdA	Analogue:U5P		Unbound	Unbound	Unbound	Unbound
1ucgA	Unbound		Unbound	Unbound	Unbound	Unbound
1ucgB	Unbound		Unbound	Unbound	Unbound	Unbound
1v9hA	Analogue:U5P		Unbound	Unbound	Unbound	Unbound
1dixA	Unbound		Unbound	Unbound	Unbound	Unbound
1vczA	Analogue:5GP_295-5GP_296		Unbound	Unbound	Unbound	Unbound
1vd1A	Unbound		Unbound	Unbound	Unbound	Unbound
1vd3A	Unbound		Unbound	Unbound	Unbound	Analogue:U2P

Reference for Active-site residues
resource	references	E.C.

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

1bolA	HIS 46;HIS 104;GLU 105;LYS 108;HIS 109
1bk7A	HIS 34;HIS 83;GLU 84;LYS 87;HIS 88
1j1fA	HIS 34;HIS 83;GLU 84;LYS 87;HIS 88
1j1gA	HIS 34;HIS 83;GLU 84;LYS 87;HIS 88
1ucaA	HIS 34;HIS 83;GLU 84;LYS 87;HIS 88
1uccA	HIS 34;HIS 83;GLU 84;LYS 87;HIS 88
1ucdA	HIS 34;HIS 83;GLU 84;LYS 87;HIS 88
1ucgA	HIS 34;HIS 83;GLU 84;LYS 87;HIS 88
1ucgB	HIS 34;HIS 83;GLU 84;LYS 87;HIS 88
1v9hA	HIS 34;HIS 83;GLU 84;LYS 87;HIS 88
1dixA	HIS 39;HIS 92;GLU 93;LYS 96;HIS 97
1vczA	HIS 39;HIS 92;GLU 93;LYS 96;HIS 97
1vd1A	HIS 39;HIS 92;GLU 93;LYS 96;HIS 97
1vd3A	HIS 39;HIS 92;GLU 93;LYS 96;HIS 97

References for Catalytic Mechanism
References	Sections	No. of steps in catalysis
[3]	p.317
[4]	p.122, Fig.3, Fig.7	2
[6]	p.863-866
[8]

References
[1]
Resource
Comments	X-ray crystallography (2.5 Angstroms)
Medline ID	92339548
PubMed ID	1633875
Journal	FEBS Lett
Year	1992
Volume	306
Pages	189-92
Authors	Kurihara H, Mitsui Y, Ohgi K, Irie M, Mizuno H, Nakamura KT
Title	Crystal and molecular structure of RNase Rh, a new class of microbial ribonuclease from Rhizopus niveus.
Related PDB
Related UniProtKB	P08056
[2]
Resource
Comments
Medline ID
PubMed ID	8218254
Journal	Biochemistry
Year	1993
Volume	32
Pages	11825-37
Authors	Nonaka T, Nakamura KT, Uesugi S, Ikehara M, Irie M, Mitsui Y
Title	Crystal structure of ribonuclease Ms (as a ribonuclease T1 homologue) complexed with a guanylyl-3',5'-cytidine analogue.
Related PDB
Related UniProtKB
[3]
Resource
Comments	X-ray crystallography (2.0 Angstroms)
Medline ID
PubMed ID	8551522
Journal	J Mol Biol
Year	1996
Volume	255
Pages	310-20
Authors	Kurihara H, Nonaka T, Mitsui Y, Ohgi K, Irie M, Nakamura KT
Title	The crystal structure of ribonuclease Rh from Rhizopus niveus at 2.0 A resolution.
Related PDB	1bol
Related UniProtKB
[4]
Resource
Comments
Medline ID
PubMed ID
Journal	In "Ribonucleases: Structures and Functions"
Year	1997
Volume	(Academic Press, New York)
Pages	101-130
Authors	Irie M. (D'Alessio G, Riordan J.)
Title	RNase T1/RNase T2 family RNases.
Related PDB
Related UniProtKB
[5]
Resource
Comments	X-ray crystallography (1.75 Angstroms)
Medline ID
PubMed ID	10446375
Journal	Biochim Biophys Acta
Year	1999
Volume	1433
Pages	253-60
Authors	Nakagawa A, Tanaka I, Sakai R, Nakashima T, Funatsu G, Kimura M
Title	Crystal structure of a ribonuclease from the seeds of bitter gourd (Momordica charantia) at 1.75 A resolution.
Related PDB	1bk7
Related UniProtKB
[6]
Resource
Comments	X-ray crystallography (1.65 Angstroms)
Medline ID
PubMed ID	10801354
Journal	J Mol Biol
Year	2000
Volume	298
Pages	859-73
Authors	Tanaka N, Arai J, Inokuchi N, Koyama T, Ohgi K, Irie M, Nakamura KT
Title	Crystal structure of a plant ribonuclease, RNase LE.
Related PDB	1dix
Related UniProtKB
[7]
Resource
Comments
Medline ID
PubMed ID	10964705
Journal	Biochem Biophys Res Commun
Year	2000
Volume	275
Pages	572-6
Authors	Suzuki A, Yao M, Tanaka I, Numata T, Kikukawa S, Yamasaki N, Kimura M
Title	Crystal structures of the ribonuclease MC1 from bitter gourd seeds, complexed with 2'-UMP or 3'-UMP, reveal structural basis for uridine specificity.
Related PDB	1uca 1ucc
Related UniProtKB
[8]
Resource
Comments
Medline ID
PubMed ID	11577107
Journal	J Biol Chem
Year	2001
Volume	276
Pages	45261-9
Authors	Matsuura T, Sakai H, Unno M, Ida K, Sato M, Sakiyama F, Norioka S
Title	Crystal structure at 1.5-A resolution of Pyrus pyrifolia pistil ribonuclease responsible for gametophytic self-incompatibility.
Related PDB	1iqq
Related UniProtKB
[9]
Resource
Comments
Medline ID
PubMed ID	12731868
Journal	Biochemistry
Year	2003
Volume	42
Pages	5270-8
Authors	Numata T, Suzuki A, Kakuta Y, Kimura K, Yao M, Tanaka I, Yoshida Y, Ueda T, Kimura M
Title	Crystal structures of the ribonuclease MC1 mutants N71T and N71S in complex with 5'-GMP: structural basis for alterations in substrate specificity.
Related PDB	1j1f 1j1g 1ucg
Related UniProtKB
[10]
Resource
Comments
Medline ID
PubMed ID	15322360
Journal	Biosci Biotechnol Biochem
Year	2004
Volume	68
Pages	1748-57
Authors	Kimura K, Numata T, Kakuta Y, Kimura M
Title	Amino acids conserved at the C-terminal half of the ribonuclease T2 family contribute to protein stability of the enzymes.
Related PDB	1v9h
Related UniProtKB

Comments
Accoriding to the literature [3], reaction mechanism is described as follows: His109 (1bol) accepts the proton form the 2'-OH group of the substrate in the first reaction step, whereas His46 is supposed to act as a general acid in the first step. Glu105 (1bol) seems to play a more important role than just adjusting the pKa value of the neighboring histidine residues [3]. The paper [6] suggests the corresponding glutamic acid is catalytically crucial and probably polarize the P=O bond or stabilize a pentacovalent intermediate together with the catalytic histidine residues. Since the pentacovalent intermediate should carry excess negative charge, it is reasonable to say that the non-ionized glutamic acid residue is appropriate for enhancing the catalysis. Moreover, according to the literature [4], [6] & [8], His104, Lys108 and protonated form of Glu105 stabilizes the pentacovalent intermediate in the transition state. Taken together, the catalytic reaction proceeds as follows: (1) His109 acts as a general base to activate 2'-OH of RNA substrate. (2) The activated 2'-hydroxyl oxygen makes a nucleophilic attack on the phosphoryl atom of the phosphodiester group, leading to pentacovalent transition state. The transition state must be stabilized by His104, Glu105 and Lys108. (3) The reaction leads to formation of 2',3'-cyclic nucleotide, and leaving 5'-OH group of the next nucleotide. Here, His46 acts as a general acid to protonate the leaving 5'-hydroxyl oxygen. (4) Probably His46 now acts as a general base to activate a water, which will complete the hydrolysis, along with the stabilizers (His104, Glu105 and Lys108). Meanwhile, His109 acts as a general acid to protonate the leaving 2'-oxygen atom.

Comments

Accoriding to the literature [3], reaction mechanism is described as follows:
His109 (1bol) accepts the proton form the 2'-OH group of the substrate in the first reaction step, whereas His46 is supposed to act as a general acid in the first step.
Glu105 (1bol) seems to play a more important role than just adjusting the pKa value of the neighboring histidine residues [3]. The paper [6] suggests the corresponding glutamic acid is catalytically crucial and probably polarize the P=O bond or stabilize a pentacovalent intermediate together with the catalytic histidine residues. Since the pentacovalent intermediate should carry excess negative charge, it is reasonable to say that the non-ionized glutamic acid residue is appropriate for enhancing the catalysis.
Moreover, according to the literature [4], [6] & [8], His104, Lys108 and protonated form of Glu105 stabilizes the pentacovalent intermediate in the transition state.
Taken together, the catalytic reaction proceeds as follows:
(1) His109 acts as a general base to activate 2'-OH of RNA substrate.
(2) The activated 2'-hydroxyl oxygen makes a nucleophilic attack on the phosphoryl atom of the phosphodiester group, leading to pentacovalent transition state. The transition state must be stabilized by His104, Glu105 and Lys108.
(3) The reaction leads to formation of 2',3'-cyclic nucleotide, and leaving 5'-OH group of the next nucleotide. Here, His46 acts as a general acid to protonate the leaving 5'-hydroxyl oxygen.
(4) Probably His46 now acts as a general base to activate a water, which will complete the hydrolysis, along with the stabilizers (His104, Glu105 and Lys108). Meanwhile, His109 acts as a general acid to protonate the leaving 2'-oxygen atom.

Created	Updated
2002-07-01	2010-02-04