This enzyme belongs to the family of isomerases, specifically those intramolecular oxidoreductases interconverting aldoses and ketoses. The systematic name of this enzyme class is N-(5-phospho-beta-D-ribosyl)anthranilate aldose-ketose-isomerase. Other names in common use include:
PRA isomerase,
PRAI,
IGPS:PRAI (indole-3-glycerol-phosphate,
synthetase/N-5'-phosphoribosylanthranilate isomerase complex), and
Phosphoribosylanthranilate isomerase is one of the many enzymes within the biosynthesis pathway of tryptophan (an essential amino acid). The upstream* pathway substrates and intermediates are shown below (Fig. 2).
As seen in Fig. 3, N-(5'-phosphoribosyl)-anthranilate via this enzyme is converted into 1-(o-carboxyphenylamino)-1-deoxribulose 5-phosphate. As the name phosphoribosylanthranilate isomerase suggests, it functions as an isomerase, rearranging the parts of the molecule without adding or removing molecules or atoms.
The reaction seen in Fig. 3, is an intramolecular redox (reduction-oxidation) reaction.[5] Its first step involves a proton transfer. This product intermediate, an enolamine, is fluorescent, which is useful for kinetic studies
within this pathway.[5] However, this product is unstable, and quickly isomerases into an α-amino keto.
Fig. 2: Upstream* Pathway of Tryptophan Synthesis
Fig 3: Enzyme Isomerase Reaction
Fig. 4: Downstream* Pathway of Tryptophan Synthesis
Note: Upstream/Downstream are relative to the compounds/molecules directly involved in phosphoribosylanthranilate isomerase reaction
Kinetics
Michaelis–Menten kinetics data, is given in the table below for PRAI and indole-glycerol-phosphate synthase (IGPS, EC 4.1.1.48).[6]
Table 1: Kinetic Data
Enzyme
Temperature (°C)
Km
(μM)
kcat
(1/sec)
tPRAI
25
0.280
3.7
45
0.390
13.5
60
0.730
38.5
80
1.030
116.8
tIGPS
25
0.006
0.11
45
0.014
0.75
60
0.053
3.24
80
0.123
15.4
Structure
Fig 6: Structure of N-(5'-phosphoribosyl) anthranilate isomerase from Pyrococcus furiosus
Depending on the microorganism PRAI's structure can vary between a mono-functional enzyme (monomeric and labile) or a stable bi-functional dimeric enzyme. Within Saccharomyces cerevisiae, Bacillus subtilis, Pseudomonas putida, and Acinetobacter calcoaceticus the enzyme is monmeric.[7] In contrast, in hyperthermophileThermotoga maritima,Escherichia coli (Fig. 5), Salmonella typhimurium, and Aerobacter aerogenes, and Serratia marcescens, it is a bi-functional enzyme with indoleglycerol phosphate synthase as the paired enzyme.[8]
The crystal structure has been characterized for a variety of the above listed microorganisms. The known 2.0 A structure of PRAI from Pyrococcus furiosus shows that tPRAI has a TIM-barrel fold (Fig. 6). PRAI derived from Thermococcus kodakaraensis also expresses a similar TIM-barrel fold structure.[7] The subunits of tPRAI associate via the N-terminal faces of their central beta-barrels. Two long, symmetry-related loops that protrude reciprocally into cavities of the other subunit provide for multiple hydrophobic interactions. Moreover, the side chains of the N-terminal methionines and the C-terminal leucines of both subunits are immobilized in a hydrophobic cluster, and the number of salt bridges is increased in tPRAI. These features appear to be mainly responsible for the high thermostability of tPRAI.[9]
Fig 5: Three dimensional structure of the bi-functional PRAI: IGPS enzyme from E. Coli Fig 7: IGPS (purple), shared (orange), and PRAI (turquoise) reaction domains
The bi-functional version of this enzyme isolated from E. Coli (Fig. 5) performs two steps within the Tryptophan pathway. Referencing Fig. 7, the N-terminal catalyzes the IGPS reaction (residues ~1–289 purple), and the C-terminal domain performs the PRAI reaction (residues ~158–452 turquoise). Although these domains overlap (orange), the active sites are not overlapping, and studies have shown that mono-functional enzymes composing of these two domains are still able to produce a functional tryptophan bio-synthetic pathway.[10]
The βα loops are responsible for the activity of this enzyme, and the αβ loops are involved in the protein's stability.[8]
More details on the discovery of this enzyme's structure can be found in Willmann's paper.[11]
Specifically, for phosphoribosyl anthranilate isomerase, TkTrpF, from Thermococcus kodakaraensis. The active site for the Amadori rearrangement, was determined to be Cys8 (acting as the general base) and Asp135 (as the general acid).[12]
Inhibitors
An enzyme inhibitor[13] is molecule that binds to an enzyme that therefore decreases the activity of the protein. The following molecules have been shown to inhibit PRAI activity:
Reduced 1-(2-carboxyphenylamino )-1-deoxy-D-ribulose 5-phosphate [5, 6,8); Indoleglycerol phosphate (8); Indolepropanol phosphate (8); MnCI2 CoCI2 [16); CuS04 (16); More (chemically synthesized N-(5-phospho-betaD-ribosyl)anthranilate contains inhibitors, but not if it is generated by anthranilate phosphoribosyltransferase)
A list of genes encoding for PRAI can also be found on KEGG Enzyme database.[15]
Related Research Articles
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The enzyme anthranilate synthase catalyzes the chemical reaction
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References
↑ Creighton TE, Yanofsky C (1970). "Chorismate to tryptophan (Escherichia coli)—anthranilate synthetase, PR transferase, PRA isomerase, InGP synthetase, tryptophan synthetase". Metabolism of Amino Acids and Amines Part A. Methods in Enzymology. Vol.17A. pp.365–380. doi:10.1016/0076-6879(71)17215-1. ISBN9780121818746.
1 2 Hommel U, Eberhard M, Kirschner K (April 1995). "Phosphoribosyl anthranilate isomerase catalyzes a reversible amadori reaction". Biochemistry. 34 (16): 5429–39. doi:10.1021/bi00016a014. PMID7727401.
↑ Sterner R, Merz A, Thoma R, Kirschner K (2001). "Phosphoribosylanthranilate isomerase and indoleglycerol-phosphate synthase: Tryptophan biosynthetic enzymes from Thermotoga maritima". Hyperthermophilic enzymes Part B. Methods in Enzymology. Vol.331. pp.270–80. doi:10.1016/S0076-6879(01)31064-9. ISBN9780121822323. PMID11265469.
↑ Hennig M, Sterner R, Kirschner K, Jansonius JN (May 1997). "Crystal structure at 2.0 A resolution of phosphoribosyl anthranilate isomerase from the hyperthermophile Thermotoga maritima: possible determinants of protein stability". Biochemistry. 36 (20): 6009–16. doi:10.1021/bi962718q. PMID9166771.
↑ Eberhard M, Tsai-Pflugfelder M, Bolewska K, Hommel U, Kirschner K (April 1995). "Indoleglycerol phosphate synthase-phosphoribosyl anthranilate isomerase: comparison of the bifunctional enzyme from Escherichia coli with engineered monofunctional domains". Biochemistry. 34 (16): 5419–28. doi:10.1021/bi00016a013. PMID7727400.
↑ PDB: 1PII; Wilmanns M, Priestle JP, Niermann T, Jansonius JN (January 1992). "Three-dimensional structure of the bifunctional enzyme phosphoribosylanthranilate isomerase: indoleglycerolphosphate synthase from Escherichia coli refined at 2.0 A resolution". Journal of Molecular Biology. 223 (2): 477–507. doi:10.1016/0022-2836(92)90665-7. PMID1738159.
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Fig. 2: Upstream* Pathway of Tryptophan Synthesis
Fig 3: Enzyme Isomerase Reaction
Fig. 4: Downstream* Pathway of Tryptophan Synthesis
