Amidophosphoribosyltransferase

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Phosphoribosyl pyrophosphate amidotransferase
ATase crystal structure.png
Structure of the ATase tetramer, generated from 1ECB.[1]
Identifiers
Symbols PPAT ; ATASE; GPAT; PRAT
External IDs OMIM172450 MGI2387203 HomoloGene68272 GeneCards: PPAT Gene
EC number 2.4.2.14
Orthologs
Species Human Mouse
Entrez 5471 231327
Ensembl ENSG00000128059 ENSMUSG00000029246
UniProt Q06203 Q3UGU3
RefSeq (mRNA) NM_002703 NM_172146
RefSeq (protein) NP_002694 NP_742158
Location (UCSC) Chr 4:
57.26 – 57.3 Mb
Chr 5:
76.91 – 76.95 Mb
PubMed search [1] [2]

Amidophosphoribosyltransferase (ATase), also known as glutamine phosphoribosylpyrophosphate amidotransferase (GPAT), is an enzyme responsible for catalyzing the conversion of 5-phosphoribosyl-1-pyrophosphate (PRPP) into 5-phosphoribosyl-1-amine (PRA), using the ammonia group from a glutamine side-chain. This is the committing step in de novo purine synthesis. In humans it is encoded by the PPAT (phosphoribosyl pyrophosphate amidotransferase) gene.[2][3] ATase is a member of the purine/pyrimidine phosphoribosyltransferase family.

Structure and function[edit]

amidophosphoribosyltransferase
Identifiers
EC number 2.4.2.14
CAS number 9031-82-7
Databases
IntEnz IntEnz view
BRENDA BRENDA entry
ExPASy NiceZyme view
KEGG KEGG entry
MetaCyc metabolic pathway
PRIAM profile
PDB structures RCSB PDB PDBe PDBsum
Gene Ontology AmiGO / EGO

The enzyme comprises of two domains: a glutaminase domain that produces ammonia from glutamine by hydrolysis and a phosphoribosyltransferase domain that binds the ammonia to ribose-5-phosphate.[4] Coordination between the two active sites of enzyme give it special complexity.

The glutaminase domain is homologous to other N-terminal nucleophile (Ntn) hydrolases [4] such as carbamoyl phosphate synthetase (CPSase). Nine invariant residues among the sequences of all Ntn amidotransferases play key catalytic, subtrate binding or structural roles. A terminal cysteine residue acts as the nucleophile in the first part of the reaction. [4][5] The free N terminus is essential to the protonation of the leaving group in the hydrolitic reaction, in this case ammonia. Another key aspect of the catalytic site is an oxyanion hole which catalizes the reaction intermediate, as shown in the mechanism below.[6]

The PRTase domain is homologous to many other PRTases involved in the purine nucleotide synthesis and salvage pathways. All PRTases involve the displacement of pyrophosphate in PRPP by a variety of nucleophiles.[7] ATase is the only PRTase that has ammonia as a nucleophile.[4] Pyrophosphate from PRPP is an excellent leaving group, so little chemical assitance is needed to promote catalysis. Rather, the primary function of the enzyme appears to be bringing the reactants together appropriately and preventing the wrong reaction, such as hydrolysis.[4]

Besides having their respective catalytic abilities, the two domains also coordinate with one another to ensure that all the ammonia produced from glutamine is transferred to PRPP and no other nucleophile than ammonia attacks PRPP. This is achieved mainly by blocking formation of ammonia until PRPP is bound and channeling the ammonia to the PRTase active site.[4]

Initial activation of the enzyme by PRPP is caused by a conformational change in a "glutamine loop", which repositions to be able to accept glutamine. This results in a 200-fold higher Km value for glutamine binding[8] Once glutamine has bound to the active site, further conformational changes bring the site into the enzyme, making it inaccessible. [4]

These conformational changes also result in the formation of a 20 Å long ammonia channel, one of the most striking features of this enzyme. This channel lacks any hydrogen bonding sites, to ensure easy diffusion of ammonia from one active site to the other. This channel ensures ammonia released from glutamine reaches the PRTase catalytic site, and it differs from the channel in CPSase[9] in that it is hydrophobic rather than polar, and transient rather than permanent. [4]

Reaction mechanism[edit]

Arrow pushing mechanism for the reaction catalyzed by ATase. Ammonia is liberated in the third step, which goes on to the second half of the mechanism.[6]
Arrow pushing mechanism for the reaction catalyzed by ATase. The ammonia liberated in the first half of the reaction replaces pyrophosphate in PRPP, yielding phosphoribosylamine. The tyrosine residue stabilizes the intermediate and allows the reaction to occur.[6]

The overall reaction catalyzed by ATase is the following:

PRPP + glutaminePRA + glutamate

Within the enzyme, the reaction is broken down into two half reactions that occur at different active sites:

glutamineNH
3
+ glutamate
PRPP + NH
3
PRA

The first part of the mechanism releases an ammonia group from glutamine by hydrolysis, which is then transferred to a second active site via a 20 Å channel, where it then binds to PRPP to form PRA.

Regulation[edit]

In an example of feedback inhibition, ATase is inhibited mainly by the end-products of the purine synthesis pathway, AMP, GMP, ADP, and GDP.[4] Each enzyme subunit from the homotetramer has two binding sites for these inhibitors. The allosteric (A) site overlaps with the site for the ribose-5-phosphate of PRPP, while the catalytic (C) site overlaps with the site for the pyrophosphate of PRPP.[4] The binding of specific nucleotide pairs to the two sites results in synergistic inhibition stronger than additive inhibition.[4][10][11] Inhibition occurs via a structural change in the enzyme where the flexible glutamine loop gets locked in an open position, preventing the binding of PRPP.[4]

Interactive pathway map[edit]

Click on genes, proteins and metabolites below to link to respective articles. [§ 1]

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FluoropyrimidineActivity_WP1601 go to article go to article go to article go to pathway article go to pathway article go to article go to article go to article go to article go to article go to article go to article go to article go to article go to PubChem Compound go to article go to article go to article go to article go to article go to article go to article go to article go to article go to article go to article go to article go to article go to article go to article go to article go to article go to article go to pathway article go to pathway article go to article go to article go to article go to article go to article go to WikiPathways go to article go to article go to article go to article go to article go to article go to article go to article go to article
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FluoropyrimidineActivity_WP1601 go to article go to article go to article go to pathway article go to pathway article go to article go to article go to article go to article go to article go to article go to article go to article go to article go to PubChem Compound go to article go to article go to article go to article go to article go to article go to article go to article go to article go to article go to article go to article go to article go to article go to article go to article go to article go to article go to pathway article go to pathway article go to article go to article go to article go to article go to article go to WikiPathways go to article go to article go to article go to article go to article go to article go to article go to article go to article
|{{{bSize}}}px|alt=Fluorouracil (5-FU) Activity edit|]]
Fluorouracil (5-FU) Activity edit
  1. ^ The interactive pathway map can be edited at WikiPathways: "FluoropyrimidineActivity_WP1601". 

Gallery[edit]

References[edit]

  1. ^ Krahn JM, Kim JH, Burns MR, Parry RJ, Zalkin H, Smith JL (Sep 1997). "Coupled formation of an amidotransferase interdomain ammonia channel and a phosphoribosyltransferase active site". Biochemistry 36 (37): 11061–11068. doi:10.1021/bi9714114. PMID 9333323. 
  2. ^ "Entrez Gene: phosphoribosyl pyrophosphate amidotransferase". 
  3. ^ Brayton KA, Chen Z, Zhou G, Nagy PL, Gavalas A, Trent JM et al. (Feb 1994). "Two genes for de novo purine nucleotide synthesis on human chromosome 4 are closely linked and divergently transcribed". The Journal of Biological Chemistry 269 (7): 5313–21. PMID 8106516. 
  4. ^ a b c d e f g h i j k l Smith JL (Dec 1998). "Glutamine PRPP amidotransferase: snapshots of an enzyme in action". Current Opinion in Structural Biology 8 (6): 686–94. doi:10.1016/s0959-440x(98)80087-0. PMID 9914248. 
  5. ^ Smith JL, Zaluzec EJ, Wery JP, Niu L, Switzer RL, Zalkin H et al. (Jun 1994). "Structure of the allosteric regulatory enzyme of purine biosynthesis". Science 264 (5164): 1427–1433. doi:10.1126/science.8197456. PMID 8197456. 
  6. ^ a b c "Overview for MACiE Entry M0214". EMBL-EBI. 
  7. ^ Musick WD (1981). "Structural features of the phosphoribosyltransferases and their relationship to the human deficiency disorders of purine and pyrimidine metabolism". CRC Critical Reviews in Biochemistry 11 (1): 1–34. doi:10.3109/10409238109108698. PMID 7030616. 
  8. ^ Kim JH, Krahn JM, Tomchick DR, Smith JL, Zalkin H (Jun 1996). "Structure and function of the glutamine phosphoribosylpyrophosphate amidotransferase glutamine site and communication with the phosphoribosylpyrophosphate site". The Journal of Biological Chemistry 271 (26): 15549–15557. PMID 8663035. 
  9. ^ Thoden JB, Holden HM, Wesenberg G, Raushel FM, Rayment I (May 1997). "Structure of carbamoyl phosphate synthetase: a journey of 96 A from substrate to product". Biochemistry 36 (21): 6305–6316. doi:10.1021/bi970503q. PMID 9174345. 
  10. ^ Chen S, Tomchick DR, Wolle D, Hu P, Smith JL, Switzer RL et al. (Sep 1997). "Mechanism of the synergistic end-product regulation of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase by nucleotides". Biochemistry 36 (35): 10718–10726. doi:10.1021/bi9711893. PMID 9271502. 
  11. ^ Zhou G, Smith JL, Zalkin H (Mar 1994). "Binding of purine nucleotides to two regulatory sites results in synergistic feedback inhibition of glutamine 5-phosphoribosylpyrophosphate amidotransferase". The Journal of Biological Chemistry 269 (9): 6784–6789. PMID 8120039. 

Further reading[edit]

External links[edit]

This article incorporates text from the United States National Library of Medicine, which is in the public domain.


Category:EC 2.4.2