Receptor protein-tyrosine kinase

 

Phosphorylated insulin receptor tyrosine kinase from Homo sapiens catalyses the phosphorylation of Tyr on intracellular proteins that are related to the STATK pathway. The phosphoryl group is transferred from ATP to form ADP. On carrying out this function, the enzyme activates the signalling cascade inside the cell. Insulin receptor tyrosine kinase is thought to have a restricted range of protein substrates, with a consensus YMXM phosphorylation motif having been defined [PMID:17085043].

There are two alternative mechanisms suggested for this enzyme, and there is still some debate as to whether this enzyme works via the associative (SN2-type) mechanism or the dissociative (SN1-type) mechanism. However, experimental and QM/MM studies, as well as crystallographic evidence, seems to support the dissociative mechanism [PMID:17085043, PMID:16023488, PMID:19334696 and Pichierri et al.].

 

Reference Protein and Structure

Sequence
P06213 UniProt (2.7.10.1) IPR016246 (Sequence Homologues) (PDB Homologues)
Biological species
Homo sapiens (Human) Uniprot
PDB
1ir3 - PHOSPHORYLATED INSULIN RECEPTOR TYROSINE KINASE IN COMPLEX WITH PEPTIDE SUBSTRATE AND ATP ANALOG (1.9 Å) PDBe PDBsum 1ir3
Catalytic CATH Domains
1.10.510.10 CATHdb (see all for 1ir3)
Cofactors
Magnesium(2+) (2) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:2.7.10.1)

ATP(4-)
CHEBI:30616ChEBI
+
L-tyrosine residue
CHEBI:46858ChEBI
→
ADP(3-)
CHEBI:456216ChEBI
+
L-tyrosine-O-phosphate(2-) residue
CHEBI:82620ChEBI
+
hydron
CHEBI:15378ChEBI
Alternative enzyme names: CDw135, Cek1, Cek10, AATK, AATYK, AATYK2, AATYK3, ACH, ALK, Anaplastic lymphoma kinase, ARK, ATP:protein-tyrosine O-phosphotransferase, AXL, Bek, Bfgfr, BRT, Bsk, C-FMS, CAK, CCK4, CD115, CD135, Cek11, Cek2, Cek3, Cek5, Cek6, Cek7, CFD1, CKIT, CSF1R, DAlk, DDR1, DDR2, Dek, DKFZp434C1418, Drosophila Eph kinase, DRT, DTK, Ebk, ECK, EDDR1, Eek, EGFR, Ehk2, Ehk3, Elk, EPH, EPHA1, EPHA2, EPHA6, EPHA7, EPHA8, EPHB1, EPHB2, EPHB3, EPHB4, EphB5, Ephrin-B3 receptor tyrosine kinase, EPHT, EPHT2, EPHT3, EPHX, ERBB, ERBB1, ERBB2, ERBB3, ERBB4, ERK, Eyk, FGFR1, FGFR2, FGFR3, FGFR4, FLG, FLK1, FLK2, FLT1, FLT2, FLT3, FLT4, FMS, Fv2, HBGFR, HEK11, HEK2, HEK3, HEK5, HEK6, HEP, HER2, HER3, HER4, HGFR, HSCR1, HTK, IGF1R, INSR, INSRR, Insulin receptor protein-tyrosine kinase, IR, IRR, JTK12, JTK13, JTK14, JWS, K-SAM, KDR, KGFR, KIA0641, KIAA1079, KIAA1459, Kil, Kin15, Kin16, KIT, KLG, LTK, MCF3, Mdk1, Mdk2, Mdk5, MEhk1, MEN2A/B, Mep, MER, MERTK, MET, Mlk1, Mlk2, Mrk, MST1R, MTC1, MUSK, Myk1, N-SAM, NEP, NET, Neu, Neurite outgrowth regulating kinase, NGL, NOK, Nork, Novel oncogene with kinase-domain, Nsk2, NTRK1, NTRK2, NTRK3, NTRK4, NTRKR1, NTRKR2, NTRKR3, Nuk, NYK, PCL, PDGFR, PDGFRA, PDGFRB, PHB6, Protein-tyrosine kinase, Protein tyrosine kinase, PTK, PTK3, PTK7, Receptor protein tyrosine kinase, RET, RON, ROR1, ROR2, ROS1, RSE, RTK, RYK, SEA, Sek2, Sek3, Sek4, Sfr, SKY, STK, STK1, TEK, TIE, TIE1, TIE2, TIF, TKT, TRK, TRKA, TRKB, TRKC, TRKE, TYK1, TYRO10, Tyro11, TYRO3, Tyro5, Tyro6, TYRO7, UFO, VEGFR1, VEGFR2, VEGFR3, Vik, YK1, Yrk,

Enzyme Mechanism

Introduction

The entry represents the proposed dissociative (SN1-type) mechanism. Here a metaphosphate anion is eliminated from the ATP substrate in an intramolecular elimination. This extremely reactive intermediate then attacks the protein tyrosine substrate in an addition reaction with concomittant deprotonation of the hydroxyl by the metaphosphate anion.

Catalytic Residues Roles

UniProt PDB* (1ir3)
Asp1177, Asn1164 Asp1150(173)A, Asn1137(160)A Form Mg binding site metal ligand
Asp1159 Asp1132(155)A Activates the substrate tyrosine, increasing its nucleophilicty. increase nucleophilicity, steric role
Arg1163 Arg1136(159)A Hydrogen bonds to the negatively charged oxygen of the gamma phosphate, stabilising the transition state. increase electrophilicity, promote heterolysis, electrostatic stabiliser
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

proton transfer, heterolysis, overall reactant used, dephosphorylation, intermediate formation, overall product formed, rate-determining step, bimolecular nucleophilic addition, intermediate terminated, native state of enzyme regenerated

References

  1. Pichierri F et al. (2003), Theochem, 622, 257-267. Mechanism of tyrosine phosphorylation catalyzed by the insulin receptor tyrosine kinase: a semiempirical PM3 study. DOI:10.1016/s0166-1280(02)00651-6.
  2. Zhou B et al. (2009), J Phys Chem A, 113, 5144-5150. A computational study of the phosphorylation mechanism of the insulin receptor tyrosine kinase. DOI:10.1021/jp810827w. PMID:19334696.
  3. Bose R et al. (2006), Curr Opin Struct Biol, 16, 668-675. Protein tyrosine kinase–substrate interactions. DOI:10.1016/j.sbi.2006.10.012. PMID:17085043.
  4. Hines AC et al. (2005), Bioorg Chem, 33, 285-297. Bisubstrate analog probes for the insulin receptor protein tyrosine kinase: Molecular yardsticks for analyzing catalytic mechanism and inhibitor design. DOI:10.1016/j.bioorg.2005.02.002. PMID:16023488.

Step 1.

The gamma phosphate of the ATP substrate eliminates itself in a heterolysis, resulting in the transfer of a proton from the gamma phosphate to the beta phosphate and formation of ADP and the highly reactive metaphosphate anion.

It should be noted that whilst the metaphosphate (PO3-) ion is very reactive and not yet observed in enzymatic phosphoryl transfer reactions, there is indirect evidence of its existence through the use of AlF3 and BeF3 mimics, as well as computational studies [doi:10.1016/s0166-1280(02)00651-6].

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp1132(155)A steric role
Arg1136(159)A electrostatic stabiliser, promote heterolysis
Asn1137(160)A metal ligand
Asp1150(173)A metal ligand

Chemical Components

proton transfer, heterolysis, overall reactant used, dephosphorylation, intermediate formation, overall product formed, rate-determining step

Step 2. The protein tyrosine initiated a nucleophilic attack on the metaphosphate intermediate in an addition reaction, the resulting species then deprotonates the tyrosine phenol group to form the final product.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp1132(155)A increase nucleophilicity, steric role
Arg1136(159)A electrostatic stabiliser, increase electrophilicity
Asn1137(160)A metal ligand
Asp1150(173)A metal ligand

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition, intermediate terminated, overall product formed, overall reactant used, native state of enzyme regenerated
Download: Image, Marvin File

Step 1.

The gamma phosphate of the ATP substrate eliminates itself in a heterolysis, resulting in the transfer of a proton from the gamma phosphate to the beta phosphate and formation of ADP and the highly reactive metaphosphate anion.

It should be noted that whilst the metaphosphate (PO3-) ion is very reactive and not yet observed in enzymatic phosphoryl transfer reactions, there is indirect evidence of its existence through the use of AlF3 and BeF3 mimics, as well as computational studies [doi:10.1016/s0166-1280(02)00651-6].

Step 2. The protein tyrosine initiated a nucleophilic attack on the metaphosphate intermediate in an addition reaction, the resulting species then deprotonates the tyrosine phenol group to form the final product.

Products.

Introduction

This entry represents the associative (SN2-type) mechanism. Here Asp1132 deprotonates the substrate Tyr hydroxide, activating it for nucleophilic attack on the gamma phosphate of ATP, which forms a pentavalent transition state. The transition state is stabilised by hydrogen bonding to Arg 1136. The leaving group is protonated by the protonated Asp 1132, assisted by the interaction of Mg(II) which serves to make the oxygen lone pair more available for protonation.

Catalytic Residues Roles

UniProt PDB* (1ir3)
Asp1177, Asn1164 Asp1150(173)A, Asn1137(160)A Form Mg binding site metal ligand
Asp1159 Asp1132(155)A Acts as a general base to deprotonate the Tyr residue hydroxide of the substrate, activating it for nucleophilic attack. proton acceptor, proton donor
Arg1163 Arg1136(159)A Hydrogen bonds to the negatively charged oxygen of the gamma phosphate, stabilising the transition state. electrostatic stabiliser
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

proton transfer, overall reactant used, intermediate formation, overall product formed, bimolecular nucleophilic addition, unimolecular elimination by the conjugate base, native state of enzyme regenerated

References

  1. Pichierri F et al. (2003), Theochem, 622, 257-267. Mechanism of tyrosine phosphorylation catalyzed by the insulin receptor tyrosine kinase: a semiempirical PM3 study. DOI:10.1016/s0166-1280(02)00651-6.

Step 1. Asp11592 deprotonates the substrate Tyr hydroxide, activating it for nucleophilic attack on the gamma phosphate of ATP, which forms a pentavalent transition state. The transition state is stabilised by hydrogen bonding to Arg 1163.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg1136(159)A electrostatic stabiliser
Asn1137(160)A metal ligand
Asp1150(173)A metal ligand
Asp1132(155)A proton acceptor

Chemical Components

proton transfer, overall reactant used, intermediate formation, overall product formed, ingold: bimolecular nucleophilic addition

Step 2. The leaving group is protonated by the protonated Asp 1159, assisted by the interaction of Mg(II) which serves to make the oxygen lone pair more available for protonation.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg1136(159)A electrostatic stabiliser
Asn1137(160)A metal ligand
Asp1150(173)A metal ligand
Asp1132(155)A proton donor

Chemical Components

ingold: unimolecular elimination by the conjugate base, proton transfer, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. Asp11592 deprotonates the substrate Tyr hydroxide, activating it for nucleophilic attack on the gamma phosphate of ATP, which forms a pentavalent transition state. The transition state is stabilised by hydrogen bonding to Arg 1163.

Step 2. The leaving group is protonated by the protonated Asp 1159, assisted by the interaction of Mg(II) which serves to make the oxygen lone pair more available for protonation.

Products.

Contributors

Gemma L. Holliday, Anna Waters, Craig Porter, Charity Hornby