Low molecular weight phosphotyrosine protein phosphatase

 

Acts on tyrosine phosphorylated proteins, low-MW aryl phosphates and natural and synthetic acyl phosphates.

It is part of the PTPase superfamily. Has high homology to the Saccharomyces cerevisiae protein. Has conserved catalytic components, in particular the CXXXXXRS/T 'P loop'. The mechanism is therefore thought to be the same as that of the rest of the superfamily.

 

Reference Protein and Structure

Sequence
P11064 UniProt (3.1.3.2, 3.1.3.48) IPR002115 (Sequence Homologues) (PDB Homologues)
Biological species
Bos taurus (Cattle) Uniprot
PDB
1pnt - CRYSTAL STRUCTURE OF BOVINE HEART PHOSPHOTYROSYL PHOSPHATASE AT 2.2 ANGSTROMS RESOLUTION (2.2 Å) PDBe PDBsum 1pnt
Catalytic CATH Domains
3.40.50.2300 CATHdb (see all for 1pnt)
Click To Show Structure

Enzyme Reaction (EC:3.1.3.48)

water
CHEBI:15377ChEBI
+
L-tyrosine-O-phosphate(2-) residue
CHEBI:82620ChEBI
→
hydrogenphosphate
CHEBI:43474ChEBI
+
L-tyrosine residue
CHEBI:46858ChEBI
Alternative enzyme names: PPT-phosphatase, PTP-phosphatase, PTPase, (Phosphotyrosine)protein phosphatase, Phosphoprotein phosphatase (phosphotyrosine), Phosphotyrosine histone phosphatase, Phosphotyrosine phosphatase, Phosphotyrosine protein phosphatase, Phosphotyrosylprotein phosphatase, Protein phosphotyrosine phosphatase, Tyrosine O-phosphate phosphatase, Tyrosylprotein phosphatase,

Enzyme Mechanism

Introduction

The mechanism is thought to be primarily dissociative, with more catalytic importance placed on stabilising the negative charge on the leaving group, rather than activation of the nucleophile. Phosphoryl transfer occurs in a single step, with an inline displacement mechanism.

  1. Ser 20 hydrogen bonds to Cys 13, lowering Cys 13's pKa and stabilising a thiolate anion on this residue.
  2. Asp 132 is protonated, and acts as a general acid to protonate the substrate tyrosine residue, promoting phosphoryl transfer.
  3. The trigonal bipyramid transition state is stabilised by Arg 19.
  4. Cys 13 becomes phosphorylated. The substrate is released and water enters the active site.
  5. Asp 132 activates the water molecule by hydrogen bonding.
  6. During phosphoryl transfer, Ser 19 stabilises the growing negative charge on the thiolate of Cys 12. Arg 18 again stabilises the transition state.
  7. Inorganic phosphate is formed, with Asp 129 deprotonating the nucleophilic water to regenerate its protonated state.
There is computational evidence that suggests that, for low molecular weight PTPs, Cys 12 is protonated in the ground state, and transfers its proton to the phosphoryl group (i.e. substrate assisted catalysis) before nucleophilic attack.

Catalytic Residues Roles

UniProt PDB* (1pnt)
Cys13 Cys12A The nucleophilic active site residue that displaces the substrate from the phosphoryl group, becoming phosphorylated itself. covalent catalysis
Cys18 Cys17A Important for optimal positioning of the substrate phosphate moiety. steric role
Arg19 Arg18A Stabilises the transition states specifically, via a bidentate guanidinium-phosphoryl interaction. transition state stabiliser
Asp130 Asp129A Acts as the acid-base catalyst, protonating the substrate to encourage phosphoryl dissociation, and deprotonating the water nucleophile. proton shuttle (general acid/base)
Asn16 Asn15A Helps lower the pKa of Cys 12, making it more nucleophilic. It also stabilises the thiolate anion as a leaving group in the phosphoenzyme hydrolysis step. activator, electrostatic stabiliser
Ser20 Ser19A The hydroxyl group of Ser 19 lowers the pKa of Cys 12, making it more nucleophilic. It also stabilises the thiolate anion as a leaving group in the phosphoenzyme hydrolysis step. activator, 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

References

  1. Tabernero L et al. (2008), FEBS J, 275, 867-882. Protein tyrosine phosphatases: structure-function relationships. DOI:10.1111/j.1742-4658.2008.06251.x. PMID:18298793.
  2. He R et al. (2016), J Med Chem, 59, 9094-9106. Inhibition of Low Molecular Weight Protein Tyrosine Phosphatase by an Induced-Fit Mechanism. DOI:10.1021/acs.jmedchem.6b00993. PMID:27676368.
  3. Maccari R et al. (2012), J Med Chem, 55, 2-22. Low Molecular Weight Phosphotyrosine Protein Phosphatases as Emerging Targets for the Design of Novel Therapeutic Agents. DOI:10.1021/jm200607g. PMID:21988196.
  4. Madhurantakam C et al. (2008), Proteins, 71, 706-714. Analyzing the catalytic mechanism of MPtpA: A low molecular weight protein tyrosine phosphatase fromMycobacterium tuberculosis through site-directed mutagenesis. DOI:10.1002/prot.21816. PMID:17975835.
  5. Zhang ZY (2003), Prog Nucleic Acid Res Mol Biol, 73, 171-220. Mechanistic Studies on Protein Tyrosine Phosphatases. DOI:10.1016/s0079-6603(03)01006-7. PMID:12882518.
  6. Jackson MD et al. (2001), Chem Rev, 101, 2313-2340. Molecular Reactions of Protein PhosphatasesInsights from Structure and Chemistry. DOI:10.1021/cr000247e.
  7. Wang S et al. (2000), Biochemistry, 39, 1903-1914. Crystal Structures of a Low-Molecular Weight Protein Tyrosine Phosphatase fromSaccharomyces cerevisiaeand Its Complex with the Substratep-Nitrophenyl Phosphate†,‡. DOI:10.1021/bi991348d. PMID:10684639.
  8. Kolmodin K et al. (1999), Proteins, 36, 370-379. Mechanism of substrate dephosphorylation in low Mr protein tyrosine phosphatase. DOI:10.1002/(sici)1097-0134(19990815)36:3<370::aid-prot11>3.3.co;2-0. PMID:10409830.
  9. Zhang M et al. (1997), Biochemistry, 36, 15-23. Crystal Structure of Bovine Low Molecular Weight Phosphotyrosyl Phosphatase Complexed with the Transition State Analog Vanadate†,‡. DOI:10.1021/bi961804n. PMID:8993313.
  10. Davis JP et al. (1994), J Biol Chem, 269, 8734-8740. Kinetic and site-directed mutagenesis studies of the cysteine residues of bovine low molecular weight phosphotyrosyl protein phosphatase. PMID:8132604.
  11. Chiarugi P et al. (1992), FEBS Lett, 310, 9-12. Differential role of four cysteines on the activity of a low M(r) phosphotyrosine protein phosphatase. PMID:1526287.

Step 1.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg18A transition state stabiliser
Ser19A electrostatic stabiliser, activator
Asp129A proton shuttle (general acid/base)
Cys17A steric role
Cys12A covalent catalysis
Asn15A activator, electrostatic stabiliser

Chemical Components

Step 1.

Contributors

Gemma L. Holliday, Alex Gutteridge, Craig Porter, Jonathan T. W. Ng