EzCatDB: T00050
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DB codeT00050
RLCP classification3.133.300000.396 : Transfer
CATH domainDomain 13.90.63.10 : mRNA Capping Enzyme; Chain A, domain 1Catalytic domain
Domain 22.40.50.140 : OB fold (Dihydrolipoamide Acetyltransferase, E2P)Catalytic domain
Domain 34.10.87.10 : mRNA Capping Enzyme; ChainCatalytic domain
E.C.2.7.7.50

CATH domainRelated DB codes (homologues)
2.40.50.140 : OB fold (Dihydrolipoamide Acetyltransferase, E2P)M00220,M00186,D00291,D00294,T00254

Enzyme Name
UniProtKBKEGG

Q84424P78587
Protein namemRNA-capping enzymemRNA-capping enzyme subunit alphamRNA guanylyltransferase
mRNA capping enzyme
messenger RNA guanylyltransferase
Protein 2
SynonymsGTP--RNA guanylyltransferase
mRNA guanylyltransferase
EC 2.7.7.50
GTP--RNA guanylyltransferase
GTase
mRNA guanylyltransferase
EC 2.7.7.50
RefSeqNP_048451.1 (Protein)
NC_000852.5 (DNA/RNA sequence)

PfamPF03919 (mRNA_cap_C)
PF01331 (mRNA_cap_enzyme)
[Graphical view]
PF03919 (mRNA_cap_C)
PF01331 (mRNA_cap_enzyme)
[Graphical view]


UniProtKB:Accession NumberQ84424P78587
Entry nameMCE_PBCV1MCE1_CANAL
ActivityGTP + (5'')pp-Pur-mRNA = diphosphate + G(5'')ppp-Pur-mRNA.GTP + (5'')pp-Pur-mRNA = diphosphate + G(5'')ppp-Pur-mRNA.
SubunitMonomer.The mRNA-capping enzyme is composed of two separate chains alpha and beta, respectively a mRNA guanylyltransferase and an RNA 5''-triphosphatase.
Subcellular location
Nucleus.
CofactorMagnesium or manganese.

Compound table: links to PDB-related databases & PoSSuM

CofactorsSubstratesProductsintermediates
KEGG-idC00305C00034C00044C02100C00013C02031
CompoundMagnesiumManganeseGTP(5')ppPur-mRNAPyrophosphateG(5')pppR-RNAGMP covalently bonded to Lys82
Typedivalent metal (Ca2+, Mg2+)heavy metalamide group,amine group,nucleotidenucleic acids,phosphate group/phosphate ionphosphate group/phosphate ionamide group,amine group,nucleic acids,nucleotide
ChEBI18420
18291
35154
15996

29888


PubChem888
23930
6830

21961011
1023


               
1ckmA01UnboundUnboundUnboundUnboundUnboundUnboundUnbound
1ckmB01UnboundUnboundUnboundUnboundUnboundUnboundUnbound
1cknA01UnboundUnboundUnboundUnboundUnboundUnboundUnbound
1cknB01UnboundBound:_MNUnboundUnboundAnalogue:SO4UnboundUnbound
1ckoA01UnboundUnboundUnboundUnboundUnboundUnboundUnbound
1p16A01UnboundUnboundUnboundUnboundAnalogue:PO4 7001UnboundUnbound
1p16B01UnboundUnboundUnboundUnboundUnboundUnboundUnbound
1ckmA02UnboundUnboundUnboundUnboundUnboundUnboundUnbound
1ckmB02UnboundUnboundUnboundUnboundUnboundUnboundUnbound
1cknA02UnboundUnboundUnboundUnboundUnboundUnboundUnbound
1cknB02UnboundUnboundUnboundUnboundUnboundUnboundUnbound
1ckoA02UnboundUnboundUnboundUnboundUnboundUnboundUnbound
1p16A03UnboundUnboundUnboundUnboundUnboundUnboundUnbound
1p16B03UnboundUnboundUnboundUnboundUnboundUnboundUnbound
1ckmA03UnboundUnboundBound:GTPUnboundUnboundUnboundUnbound
1ckmB03UnboundUnboundBound:GTPUnboundUnboundUnboundUnbound
1cknA03UnboundUnboundBound:GTPUnboundUnboundUnboundUnbound
1cknB03UnboundUnboundUnboundUnboundUnboundUnboundBound:GPL
1ckoA03UnboundUnboundUnboundUnboundUnboundBound:GP3Unbound
1p16A02UnboundUnboundUnboundUnboundUnboundUnboundBound:__G
1p16B02UnboundUnboundBound:GTPUnboundUnboundUnboundUnbound

Active-site residues
resource
literature [11]
pdbCatalytic residuesModified residuescomment
           
1ckmA01ARG 106
 
 
1ckmB01ARG 106
 
 
1cknA01ARG 106
 
 
1cknB01ARG 106
 
 
1ckoA01ARG 106
 
 
1p16A01ARG  92
 
 
1p16B01ARG  92
 
 
1ckmA02LYS 236;ASP 244;ARG 295;LYS 298;ASN 302
 
 
1ckmB02LYS 236;ASP 244;ARG 295;LYS 298;ASN 302
 
 
1cknA02LYS 236;ASP 244;ARG 295;LYS 298;ASN 302
 
 
1cknB02LYS 236;ASP 244;ARG 295;LYS 298;ASN 302
 
 
1ckoA02LYS 236;ASP 244;ARG 295;LYS 298;ASN 302
 
 
1p16A03LYS 243;ASP 251;ARG 342;LYS 345;ASN 349
 
 
1p16B03LYS 243;ASP 251;ARG 342;LYS 345;ASN 349
 
 
1ckmA03LYS  82;ASP 213;LYS 234
 
 
1ckmB03LYS  82;ASP 213;LYS 234
 
 
1cknA03LYS  82;ASP 213;LYS 234
 
 
1cknB03       ;ASP 213;LYS 234
GPL  82
GPL guanylated lysine
1ckoA03LYS  82;ASP 213;LYS 234
 
 
1p16A02LYS  67;ASP 220;LYS 241
 
 
1p16B02LYS  67;ASP 220;LYS 241
 
 

References for Catalytic Mechanism
ReferencesSectionsNo. of steps in catalysis
[8]Fig.7, p.549-552
[10]p.9575-9576
[11]Fig.4, p.1508-1509
[15]

[16]p.1556-1558
[17]p.761-762

references
[1]
PubMed ID2159008
JournalJ Biol Chem
Year1990
Volume265
Pages7669-72
AuthorsFausnaugh J, Shatkin AJ
TitleActive site localization in a viral mRNA capping enzyme.
[2]
PubMed ID2161527
JournalProc Natl Acad Sci U S A
Year1990
Volume87
Pages4023-7
AuthorsGuo PX, Moss B
TitleInteraction and mutual stabilization of the two subunits of vaccinia virus mRNA capping enzyme coexpressed in Escherichia coli.
[3]
PubMed ID8385101
JournalJ Biol Chem
Year1993
Volume268
Pages7256-60
AuthorsCong P, Shuman S
TitleCovalent catalysis in nucleotidyl transfer. A KTDG motif essential for enzyme-GMP complex formation by mRNA capping enzyme is conserved at the active sites of RNA and DNA ligases.
[4]
PubMed ID8227060
JournalJ Biol Chem
Year1993
Volume268
Pages24986-9
AuthorsNiles EG, Christen L
TitleIdentification of the vaccinia virus mRNA guanyltransferase active site lysine.
[5]
PubMed ID8195132
JournalJ Biol Chem
Year1994
Volume269
Pages14974-81
AuthorsHigman MA, Christen LA, Niles EG
TitleThe mRNA (guanine-7-)methyltransferase domain of the vaccinia virus mRNA capping enzyme. Expression in Escherichia coli and structural and kinetic comparison to the intact capping enzyme.
[6]
PubMed ID7991582
JournalProc Natl Acad Sci U S A
Year1994
Volume91
Pages12046-50
AuthorsShuman S, Liu Y, Schwer B
TitleCovalent catalysis in nucleotidyl transfer reactions: essential motifs in Saccharomyces cerevisiae RNA capping enzyme are conserved in Schizosaccharomyces pombe and viral capping enzymes and among polynucleotide ligases.
[7]
PubMed ID7565775
JournalMol Cell Biol
Year1995
Volume15
Pages6222-31
AuthorsCong P, Shuman S
TitleMutational analysis of mRNA capping enzyme identifies amino acids involved in GTP binding, enzyme-guanylate formation, and GMP transfer to RNA.
[8]
CommentsX-RAY CRYSTALLOGRAPHY (2.5 ANGSTROMS)
Medline ID97304383
PubMed ID9160746
JournalCell
Year1997
Volume89
Pages545-53
AuthorsHakansson K, Doherty AJ, Shuman S, Wigley DB
TitleX-ray crystallography reveals a large conformational change during guanyl transfer by mRNA capping enzymes.
Related PDB1ckm,1ckn
Related UniProtKBQ84424
[9]
PubMed ID9371657
JournalJ Virol
Year1997
Volume71
Pages9837-43
AuthorsYu L, Martins A, Deng L, Shuman S
TitleStructure-function analysis of the triphosphatase component of vaccinia virus mRNA capping enzyme.
[10]
PubMed ID9275164
JournalProc Natl Acad Sci U S A
Year1997
Volume94
Pages9573-8
AuthorsWang SP, Deng L, Ho CK, Shuman S
TitlePhylogeny of mRNA capping enzymes.
[11]
CommentsX-RAY CRYSTALLOGRAPHY (3.1 ANGSTROMS) OF 11-327
Medline ID98132620
PubMed ID9465045
JournalProc Natl Acad Sci U S A
Year1998
Volume95
Pages1505-10
AuthorsHakansson K, Wigley DB
TitleStructure of a complex between a cap analogue and mRNA guanylyl transferase demonstrates the structural chemistry of RNA capping.
Related PDB1cko
Related UniProtKBQ84424
[12]
PubMed ID10454631
JournalNucleic Acids Res
Year1999
Volume27
Pages3253-8
AuthorsDoherty AJ
TitleConversion of a DNA ligase into an RNA capping enzyme.
[13]
PubMed ID11018011
JournalGenes Dev
Year2000
Volume14
Pages2435-40
AuthorsSchroeder SC, Schwer B, Shuman S, Bentley D
TitleDynamic association of capping enzymes with transcribing RNA polymerase II.
[14]
PubMed ID11463793
JournalJ Biol Chem
Year2001
Volume276
Pages36116-24
AuthorsHausmann S, Ho CK, Schwer B, Shuman S
TitleAn essential function of Saccharomyces cerevisiae RNA triphosphatase Cet1 is to stabilize RNA guanylyltransferase Ceg1 against thermal inactivation.
[15]
PubMed ID12846573
JournalBiochemistry
Year2003
Volume42
Pages8240-9
AuthorsSawaya R, Shuman S
TitleMutational analysis of the guanylyltransferase component of Mammalian mRNA capping enzyme.
[16]
PubMed ID12820968
JournalMol Cell
Year2003
Volume11
Pages1549-61
AuthorsFabrega C, Shen V, Shuman S, Lima CD
TitleStructure of an mRNA capping enzyme bound to the phosphorylated carboxy-terminal domain of RNA polymerase II.
[17]
PubMed ID15582400
JournalCurr Opin Struct Biol
Year2004
Volume14
Pages757-64
AuthorsShuman S, Lima CD
TitleThe polynucleotide ligase and RNA capping enzyme superfamily of covalent nucleotidyltransferases.

comments
According to the literature [8], [10], [11], the catalytic reaction probably proceeds as follows:
(1) Asp213 acts as a general base, which deprotonates the nucleophile, the sidechain of Lys82, to activate it. (Although the literature [11] suggests an alternative mechanism, in which alpha-phosphate of GTP may act as a general base to deprotonate Lys82 and Asp213 may only modulate it by hydrogen bonding, there some factors against the substrate-assisted mechanism. Cofactor magnesium/manganese ion might neutralize the negative charge changing the pKa of the phosphate group. Moreover, the proton transfer from Lys82 to the phosphate results in the loss of an ion pair, which will not be favored. see [11])
(2) The sidechain of Lys82 makes a nucleophilic attack on the alpha-phosphate group of GTP. This process leads to the transition state in which the transferred phosphate group goes through a pentacovalent state (SN2-like reaction). (During this process, the domains are in the closed conformation.)
(3) The transferred alpha-phosphate group is stabilized by Lys234 and Lys236 along with a cofactor magnesium/manganese ion. Meanwhile, Arg106, Arg295, Lys298 and Asn302 stabilize the leaving beta- and gamma-phosphate groups, along with Asp244, which might neutralize these positively charged residues.
(4) Phosphoamide intermediate is formed between Lys82 and GMP, whereas the leaving beta- and gamma-phosphate is released from the enzyme as a product, diphosphate.
(5) The terminal phosphate group of the second substrate, (5')ppPur-mRNA, is bound to the active site. (During this process, the enzyme is in the open conformation.)
(6) The oxygen atom of the terminal phosphate group makes a nucleophilic attack on the phosphorus atom of GMP attached to Lys82. This process leads to the transition state in which the transferred phosphate group goes through a pentacovalent state (SN2-like reaction).
(7) The transferred phosphate group is again stabilized by Lys234 and Lys236 along with a cofactor magnesium/manganese ion. Meanwhile, Arg106, Arg295, Lys298 and Asn302 stabilize the acceptor phosphate groups, along with Asp244, which might neutralize these positively charged residues.
(8) Asp213 acts as a general acid to protonate Lys82, which is finally released from the product.

createdupdated
2004-03-252009-02-26


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