{{Short description|Mammalian protein found in Homo sapiens}} {{Infobox_gene}} '''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 amine 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.<ref name="entrez">{{cite web | title = Entrez Gene: phosphoribosyl pyrophosphate amidotransferase| url = https://www.ncbi.nlm.nih.gov/gene?Db=gene&Cmd=ShowDetailView&TermToSearch=5471}}</ref><ref name="pmid8106516">{{cite journal | vauthors = Brayton KA, Chen Z, Zhou G, Nagy PL, Gavalas A, Trent JM, Deaven LL, Dixon JE, Zalkin H | title = Two genes for de novo purine nucleotide synthesis on human chromosome 4 are closely linked and divergently transcribed | journal = The Journal of Biological Chemistry | volume = 269 | issue = 7 | pages = 5313–21 | date = Feb 1994 | doi = 10.1016/S0021-9258(17)37689-5 | pmid = 8106516 | doi-access = free }}</ref> ATase is a member of the purine/pyrimidine phosphoribosyltransferase family.
== Structure and function == {{infobox enzyme | align = left | Name = amidophosphoribosyltransferase | EC_number = 2.4.2.14 | CAS_number = 9031-82-7 | GO_code = 0004044 | image = | width = | caption = }}
The enzyme consists 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.<ref name = Smith>{{cite journal | vauthors = Smith JL | title = Glutamine PRPP amidotransferase: snapshots of an enzyme in action | journal = Current Opinion in Structural Biology | volume = 8 | issue = 6 | pages = 686–94 | date = Dec 1998 | pmid = 9914248 | doi=10.1016/s0959-440x(98)80087-0}}</ref> Coordination between the two active sites of enzyme give it special complexity.
The glutaminase domain is homologous to other N-terminal nucleophile (Ntn) hydrolases<ref name = "Smith"/> such as carbamoyl phosphate synthetase (CPSase). Nine invariant residues among the sequences of all Ntn amidotransferases play key catalytic, substrate binding or structural roles. A terminal cysteine residue acts as the nucleophile in the first part of the reaction, analogous to the cysteine of a catalytic triad.<ref name = "Smith"/><ref>{{cite journal | vauthors = Smith JL, Zaluzec EJ, Wery JP, Niu L, Switzer RL, Zalkin H, Satow Y | title = Structure of the allosteric regulatory enzyme of purine biosynthesis | journal = Science | volume = 264 | issue = 5164 | pages = 1427–1433 | date = Jun 1994 | pmid = 8197456 | doi=10.1126/science.8197456| bibcode = 1994Sci...264.1427S }}</ref> The free N terminus acts as a base to activate the nucleophile and protonate the leaving group in the hydrolytic reaction, in this case ammonia. Another key aspect of the catalytic site is an oxyanion hole which catalyzes the reaction intermediate, as shown in the mechanism below.<ref name = "MACiE">{{cite web|title=Overview for MACiE Entry M0214|url=http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/MACiE/entry/getPage.pl?id=M0214|publisher=EMBL-EBI|access-date=2015-03-10|archive-date=2015-04-02|archive-url=https://web.archive.org/web/20150402155917/http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/MACiE/entry/getPage.pl?id=M0214|url-status=dead}}</ref>
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.<ref>{{cite journal | vauthors = Musick WD | title = Structural features of the phosphoribosyltransferases and their relationship to the human deficiency disorders of purine and pyrimidine metabolism | journal = CRC Critical Reviews in Biochemistry | volume = 11 | issue = 1 | pages = 1–34 | date = 1981 | pmid = 7030616 | doi=10.3109/10409238109108698}}</ref> ATase is the only PRTase that has ammonia as a nucleophile.<ref name = "Smith"/> Pyrophosphate from PRPP is an excellent leaving group, so little chemical assistance 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.<ref name = "Smith" />
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 channelling the ammonia to the PRTase active site.<ref name = "Smith"/>
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 K<sub>m</sub> value for glutamine binding<ref>{{cite journal | vauthors = Kim JH, Krahn JM, Tomchick DR, Smith JL, Zalkin H | title = Structure and function of the glutamine phosphoribosylpyrophosphate amidotransferase glutamine site and communication with the phosphoribosylpyrophosphate site | journal = The Journal of Biological Chemistry | volume = 271 | issue = 26 | pages = 15549–15557 | date = Jun 1996 | pmid = 8663035 | doi = 10.1074/jbc.271.26.15549 | doi-access = free }}</ref> Once glutamine has bound to the active site, further conformational changes bring the site into the enzyme, making it inaccessible.<ref name = "Smith"/>
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<ref>{{cite journal | vauthors = Thoden JB, Holden HM, Wesenberg G, Raushel FM, Rayment I | title = Structure of carbamoyl phosphate synthetase: a journey of 96 A from substrate to product | journal = Biochemistry | volume = 36 | issue = 21 | pages = 6305–6316 | date = May 1997 | pmid = 9174345 | doi = 10.1021/bi970503q | citeseerx = 10.1.1.512.5333 }}</ref> in that it is hydrophobic rather than polar, and transient rather than permanent.<ref name = "Smith"/>
== Reaction mechanism == {{multiple image | direction = vertical | width = 400 | image1 = Mechanism of ATase, first half.png | alt1 = Arrow pushing mechanism for the reaction catalyzed by ATase. | caption1 = First half of the catalytic mechanism of ATase occurring in the glutaminase domain active site. The catalytic cysteine performs a nucleophilic attack on the substrate to form an acyl-enzyme intermediate, which is resolved by hydrolysis. Ammonia is produced in the third step, which is used in the second half of the mechanism. | image2 = Mechanism of ATase, second half.png | alt2 = Arrow pushing mechanism for the reaction catalyzed by ATase. | caption2 = Second half of the catalytic mechanism of ATase occurring in the phosphoribosyltransferase domain active site. The ammonia liberated in the first half of the reaction replaces pyrophosphate in PRPP, yielding phosphoribosylamine. A tyrosine residue stabilizes the transition state and allows the reaction to occur. }}
The overall reaction catalyzed by ATase is the following:
: {{chem|PRPP}} + {{chem|glutamine}} → {{chem|PRA}} + {{chem|glutamate}} + PPi
Within the enzyme, the reaction is broken down into two half-reactions that occur at different active sites:
#{{chem|glutamine}} → {{chem|NH|3}} + {{chem|glutamate}} #{{chem|PRPP}} + {{chem|NH|3}} → {{chem|PRA}} + PPi
The first part of the mechanism occurs in the active site of the glutaminase domain and releases an ammonia group from glutamine by hydrolysis. The ammonia released by the first reaction is then transferred to the active site of the phosphoribosyltransferase domain via a 20 Å channel, where it then binds to PRPP to form PRA.
== Regulation == In an example of feedback inhibition, ATase is inhibited mainly by the end-products of the purine synthesis pathway, AMP, GMP, ADP, and GDP.<ref name= "Smith"/> 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.<ref name="Smith"/> The binding of specific nucleotide pairs to the two sites results in synergistic inhibition stronger than additive inhibition.<ref name="Smith"/><ref>{{cite journal | vauthors = Chen S, Tomchick DR, Wolle D, Hu P, Smith JL, Switzer RL, Zalkin H | title = Mechanism of the synergistic end-product regulation of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase by nucleotides | journal = Biochemistry | volume = 36 | issue = 35 | pages = 10718–10726 | date = Sep 1997 | pmid = 9271502 | doi = 10.1021/bi9711893 }}</ref><ref>{{cite journal | vauthors = Zhou G, Smith JL, Zalkin H | title = Binding of purine nucleotides to two regulatory sites results in synergistic feedback inhibition of glutamine 5-phosphoribosylpyrophosphate amidotransferase | journal = The Journal of Biological Chemistry | volume = 269 | issue = 9 | pages = 6784–6789 | date = Mar 1994 | doi = 10.1016/S0021-9258(17)37444-6 | pmid = 8120039 | doi-access = free }}</ref> 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.<ref name="Smith"/>
Due to the chemical lability of PRA, which has a half-life of 38 seconds at pH 7.5 and 37 °C, researchers have suggested that the compound is channeled from Amidophosphoribosyltransferase to GAR synthetase ''in vivo''.<ref name="pmid8626510">{{cite journal | vauthors = Antle VD, Liu D, McKellar BR, Caperelli CA, Hua M, Vince R | title = Substrate specificity of glycinamide ribonucleotide synthetase from chicken liver | journal = The Journal of Biological Chemistry | volume = 271 | issue = 14 | pages = 8192–5 | year = 1996 | pmid = 8626510 | doi = 10.1074/jbc.271.14.8192 | doi-access = free }}</ref>
== Interactive pathway map == {{FluoropyrimidineActivity WP1601|highlight=Amidophosphoribosyltransferase}}
== Gallery ==
<gallery> Image:Phosphoribosyl pyrophosphate.svg|PRPP Image:Phosphoribosylamine.svg|5-phosphoribosylamine </gallery>
== References == {{Reflist|33em}}
== Further reading == {{refbegin|33em}} * {{cite journal | vauthors = Iwahana H, Oka J, Mizusawa N, Kudo E, Ii S, Yoshimoto K, Holmes EW, Itakura M | title = Molecular cloning of human amidophosphoribosyltransferase | journal = Biochemical and Biophysical Research Communications | volume = 190 | issue = 1 | pages = 192–200 | date = Jan 1993 | pmid = 8380692 | doi = 10.1006/bbrc.1993.1030 | bibcode = 1993BBRC..190..192I }} * {{cite journal | vauthors = Gassmann MG, Stanzel A, Werner S | title = Growth factor-regulated expression of enzymes involved in nucleotide biosynthesis: a novel mechanism of growth factor action | journal = Oncogene | volume = 18 | issue = 48 | pages = 6667–76 | date = Nov 1999 | pmid = 10597272 | doi = 10.1038/sj.onc.1203120 | doi-access = free }} * {{cite journal | vauthors = Chen S, Nagy PL, Zalkin H | title = Role of NRF-1 in bidirectional transcription of the human GPAT-AIRC purine biosynthesis locus | journal = Nucleic Acids Research | volume = 25 | issue = 9 | pages = 1809–16 | date = May 1997 | pmid = 9108165 | pmc = 146651 | doi = 10.1093/nar/25.9.1809 }} * {{cite journal | vauthors = Stanley W, Chu EH | title = Assignment of the gene for phosphoribosylpyrophosphate amidotransferase to the pter leads to q21 region of human chromosome 4 | journal = Cytogenetics and Cell Genetics | volume = 22 | issue = 1–6 | pages = 228–31 | year = 1978 | pmid = 752480 | doi = 10.1159/000130943 }} * {{cite journal | vauthors = Maruyama K, Sugano S | title = Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides | journal = Gene | volume = 138 | issue = 1–2 | pages = 171–4 | date = Jan 1994 | pmid = 8125298 | doi = 10.1016/0378-1119(94)90802-8 }} * {{cite journal | vauthors = Bera AK, Chen S, Smith JL, Zalkin H | title = Interdomain signaling in glutamine phosphoribosylpyrophosphate amidotransferase | journal = The Journal of Biological Chemistry | volume = 274 | issue = 51 | pages = 36498–504 | date = Dec 1999 | pmid = 10593947 | doi = 10.1074/jbc.274.51.36498 | doi-access = free }} * {{cite book | vauthors = Zalkin H, Dixon JE | title = De novo purine nucleotide biosynthesis | journal = Progress in Nucleic Acid Research and Molecular Biology | volume = 42 | pages = 259–87 | year = 1992 | pmid = 1574589 | doi = 10.1016/s0079-6603(08)60578-4 | isbn = 9780125400428 }} * {{cite journal | vauthors = Suzuki Y, Yoshitomo-Nakagawa K, Maruyama K, Suyama A, Sugano S | title = Construction and characterization of a full length-enriched and a 5'-end-enriched cDNA library | journal = Gene | volume = 200 | issue = 1–2 | pages = 149–56 | date = Oct 1997 | pmid = 9373149 | doi = 10.1016/S0378-1119(97)00411-3 }} {{refend}}
== External links == * {{MeshName|Amidophosphoribosyl transferase}} * {{UCSC gene info|PPAT}}
{{Nucleotide metabolism}} {{Glycosyltransferases}} {{Enzymes}} {{Portal bar|Biology|border=no}}
{{NLM content}}
Category:EC 2.4.2