Chorismate mutase

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Chorismate mutase
Chorismate mutase with TSA bound 4.jpg
Crystal structure of chorismate mutase with a transition state analogue bound
Identifiers
EC no. 5.4.99.5
CAS no. 9068-30-8
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In enzymology, chorismate mutase (EC 5.4.99.5) is an enzyme that catalyzes the chemical reaction for the conversion of chorismate to prephenate in the pathway to the production of phenylalanine and tyrosine, also known as the shikimate pathway. Hence, this enzyme has one substrate, chorismate, and one product, prephenate. Chorismate mutase is found at a branch point in the pathway. The enzyme channels the substrate, chorismate to the biosynthesis of tyrosine and phenylalanine and away from tryptophan. [1] Its role in maintaining the balance of these aromatic amino acids in the cell is vital. [2] This is the single known example of a naturally occurring enzyme catalyzing a pericyclic reaction. [2] [nb 1] Chorismate mutase is only found in fungi, bacteria, and higher plants. Some varieties of this protein may use the morpheein model of allosteric regulation. [4]

Contents

Protein family

Chorismate mutase. Rendered from PDB 2CHS. Chorismate-mutase-pdb-2CHS.png
Chorismate mutase. Rendered from PDB 2CHS.

This enzyme belongs to the family of isomerases, specifically those intramolecular transferases that transfer functional groups. The systematic name of this enzyme class is chorismate pyruvatemutase. Chorismate mutase, also known as hydroxyphenylpyruvate synthase, participates in phenylalanine, tyrosine and tryptophan biosynthesis. [1] The structures of chorismate mutases vary in different organisms, but the majority belong to the AroQ family and are characterized by an intertwined homodimer of 3-helical subunits. Most chorismate mutases in this family look similar to that of Escherichia coli . For example, the secondary structure of the chorismate mutase of yeast is very similar to that of E. coli. Chorimate mutase in the AroQ family are more common in nature and are widely distributed among the prokaryotes. [1] For optimal function, they usually have to be accompanied by another enzyme such as prephenate dehydrogenase. These chorismate mutases are typically bifunctional enzymes, meaning they contain two catalytic capacities in the same polypeptide chain. [1] However, the chorismate mutase of eukaryotic organisms are more commonly monofunctional. There are organisms such as Bacillus subtilis whose chorismate mutase have a completely different structure and are monofunctional. These enzymes belong to the AroH family and are characterized by a trimeric α/β barrel topology. [5]

Mechanism of catalysis

The conversion of chorismate to prephenate is the first committed step in the pathway to the production of the aromatic amino acids: tyrosine and phenylalanine. The presence of chorismate mutase increases the rate of the reaction a million fold. [6] In the absence of enzyme catalysis this mechanism proceeds as a concerted, but asynchronous step and is an exergonic process. The mechanism for this transformation is formally a Claisen rearrangement, supported by the kinetic and isotopic data reported by Knowles, et al. [7]

Reaction catalyzed by chorismate mutase Chorismate Mutase Mechanism.jpg
Reaction catalyzed by chorismate mutase

E. coli and Yeast chorismate mutase have a limited sequence homology, but their active sites contain similar residues. The active site of the Yeast chorismate mutase contains Arg16, Arg157, Thr242, Glu246, Glu198, Asn194, and Lys168. The E. coli active site contains the same residues with the exception of these noted exchanges: Asp48 for Asn194, Gln88 for Glu248, and Ser84 for Thr242. In the enzyme active site, interactions between these specific residues and the substrate restrict conformational degrees of freedom, such that the entropy of activation is effectively reduced to zero, and thereby promotes catalysis. As a result, there is no formal intermediate, but rather a pseudo-diaxial chair-like transition state. Evidence for this conformation is provided by an inverse secondary kinetic isotope effect at the carbon directly attached to the hydroxyl group. [6] This seemingly unfavorable arrangement is achieved through a series of electrostatic interactions, which rotate the extended chain of chorismate into the conformation required for this concerted mechanism.

Transition state analogue in chorismate mutase active site of S. cerevisiae. Chorismate Mutase Active Site 2.jpg
Transition state analogue in chorismate mutase active site of S. cerevisiae.

An additional stabilizing factor in this enzyme-substrate complex is hydrogen bonding between the lone pair of the oxygen in the vinyl ether system and hydrogen bond donor residues. Not only does this stabilize the complex, but disruption of resonance within the vinyl ether destabilizes the ground state and reduces the energy barrier for this transformation. An alternative view is that electrostatic stabilization of the polarized transition state is of great importance in this reaction. In the chorismate mutase active site, the transition-state analog is stabilized by 12 electrostatic and hydrogen-bonding interactions. [8] This is shown in mutants of the native enzyme in which Arg90 is replaced with citrulline to demonstrate the importance of hydrogen bonding to stabilize the transition state. [9] Other work using chorismate mutase from Bacillus subtilis showed evidence that when a cation was aptly placed in the active site, the electrostatic interactions between it and the negatively charged transition state promoted catalysis. [2]

Additional studies have been done in order to support the relevance of a near attack conformer (NAC) in the reaction catalyzed by chorismate mutase. This NAC is the reactive conformation of the ground state that is directly converted to the transition state in the enzyme. Using thermodynamic integration (TI) methods, the standard free energies (ΔGN°) for NAC formation were calculated in six different environments. The data obtained suggests that effective catalysis is derived from stabilization of both the NAC and transition state. [10] However, other experimental evidence supports that the NAC effect observed is simply a result of electrostatic transition state stabilization. [11] [12]

Overall, there have been extensive studies on the exact mechanism of this reaction. However, the relative contribution of conformational constraint of the flexible substrate, specific hydrogen bonding to the transition state, and electrostatic interactions to the observed rate enhancement is still under discussion.

Notes

  1. ↑ Dimethylallyltryptophan synthase has been proposed to catalyze a Cope rearrangement, but this has yet to be proven definitively [3]

Related Research Articles

<span class="mw-page-title-main">Chymotrypsin</span> Digestive enzyme

Chymotrypsin (EC 3.4.21.1, chymotrypsins A and B, alpha-chymar ophth, avazyme, chymar, chymotest, enzeon, quimar, quimotrase, alpha-chymar, alpha-chymotrypsin A, alpha-chymotrypsin) is a digestive enzyme component of pancreatic juice acting in the duodenum, where it performs proteolysis, the breakdown of proteins and polypeptides. Chymotrypsin preferentially cleaves peptide amide bonds where the side chain of the amino acid N-terminal to the scissile amide bond (the P1 position) is a large hydrophobic amino acid (tyrosine, tryptophan, and phenylalanine). These amino acids contain an aromatic ring in their side chain that fits into a hydrophobic pocket (the S1 position) of the enzyme. It is activated in the presence of trypsin. The hydrophobic and shape complementarity between the peptide substrate P1 side chain and the enzyme S1 binding cavity accounts for the substrate specificity of this enzyme. Chymotrypsin also hydrolyzes other amide bonds in peptides at slower rates, particularly those containing leucine at the P1 position.

<span class="mw-page-title-main">Active site</span> Active region of an enzyme

In biology and biochemistry, the active site is the region of an enzyme where substrate molecules bind and undergo a chemical reaction. The active site consists of amino acid residues that form temporary bonds with the substrate, the binding site, and residues that catalyse a reaction of that substrate, the catalytic site. Although the active site occupies only ~10–20% of the volume of an enzyme, it is the most important part as it directly catalyzes the chemical reaction. It usually consists of three to four amino acids, while other amino acids within the protein are required to maintain the tertiary structure of the enzymes.

<span class="mw-page-title-main">Phenylalanine hydroxylase</span> Mammalian protein found in Homo sapiens

Phenylalanine hydroxylase. (PAH) (EC 1.14.16.1) is an enzyme that catalyzes the hydroxylation of the aromatic side-chain of phenylalanine to generate tyrosine. PAH is one of three members of the biopterin-dependent aromatic amino acid hydroxylases, a class of monooxygenase that uses tetrahydrobiopterin (BH4, a pteridine cofactor) and a non-heme iron for catalysis. During the reaction, molecular oxygen is heterolytically cleaved with sequential incorporation of one oxygen atom into BH4 and phenylalanine substrate. In humans, mutations in its encoding gene, PAH, can lead to the metabolic disorder phenylketonuria.

In biochemistry, isomerases are a general class of enzymes that convert a molecule from one isomer to another. Isomerases facilitate intramolecular rearrangements in which bonds are broken and formed. The general form of such a reaction is as follows:

<span class="mw-page-title-main">Malate dehydrogenase</span> Class of enzymes

Malate dehydrogenase (EC 1.1.1.37) (MDH) is an enzyme that reversibly catalyzes the oxidation of malate to oxaloacetate using the reduction of NAD+ to NADH. This reaction is part of many metabolic pathways, including the citric acid cycle. Other malate dehydrogenases, which have other EC numbers and catalyze other reactions oxidizing malate, have qualified names like malate dehydrogenase (NADP+).

Shikimic acid, more commonly known as its anionic form shikimate, is a cyclohexene, a cyclitol and a cyclohexanecarboxylic acid. It is an important biochemical metabolite in plants and microorganisms. Its name comes from the Japanese flower shikimi, from which it was first isolated in 1885 by Johan Fredrik Eykman. The elucidation of its structure was made nearly 50 years later.

<span class="mw-page-title-main">Prephenic acid</span> Chemical compound

Prephenic acid, commonly also known by its anionic form prephenate, is an intermediate in the biosynthesis of the aromatic amino acids phenylalanine and tyrosine, as well as of a large number of secondary metabolites of the shikimate pathway.

<span class="mw-page-title-main">Oxyanion hole</span> Pocket in the active site of an enzyme

An oxyanion hole is a pocket in the active site of an enzyme that stabilizes transition state negative charge on a deprotonated oxygen or alkoxide. The pocket typically consists of backbone amides or positively charged residues. Stabilising the transition state lowers the activation energy necessary for the reaction, and so promotes catalysis. For example, proteases such as chymotrypsin contain an oxyanion hole to stabilise the tetrahedral intermediate anion formed during proteolysis and protects substrate's negatively charged oxygen from water molecules. Additionally, it may allow for insertion or positioning of a substrate, which would suffer from steric hindrance if it could not occupy the hole. Enzymes that catalyse multi-step reactions can have multiple oxyanion holes that stabilise different transition states in the reaction.

<span class="mw-page-title-main">Enzyme catalysis</span> Catalysis of chemical reactions by specialized proteins known as enzymes

Enzyme catalysis is the increase in the rate of a process by a biological molecule, an "enzyme". Most enzymes are proteins, and most such processes are chemical reactions. Within the enzyme, generally catalysis occurs at a localized site, called the active site.

<span class="mw-page-title-main">Carboxypeptidase A</span>

Carboxypeptidase A usually refers to the pancreatic exopeptidase that hydrolyzes peptide bonds of C-terminal residues with aromatic or aliphatic side-chains. Most scientists in the field now refer to this enzyme as CPA1, and to a related pancreatic carboxypeptidase as CPA2.

<span class="mw-page-title-main">Prephenate dehydrogenase</span> Class of enzymes

Prephenate dehydrogenase is an enzyme found in the shikimate pathway, and helps catalyze the reaction from prephenate to tyrosine.

<span class="mw-page-title-main">D-lysine 5,6-aminomutase</span>

In enzymology, D-lysine 5,6-aminomutase is an enzyme that catalyzes the chemical reaction

<span class="mw-page-title-main">Phosphoenolpyruvate mutase</span> Enzyme

In enzymology, a phosphoenolpyruvate mutase is an enzyme that catalyzes the chemical reaction

<span class="mw-page-title-main">Steroid Delta-isomerase</span>

In enzymology, a steroid Δ5-isomerase is an enzyme that catalyzes the chemical reaction

<span class="mw-page-title-main">Arogenate dehydratase</span> Enzyme

Arogenate dehydratase (ADT) (EC 4.2.1.91) is an enzyme that catalyzes the chemical reaction

<span class="mw-page-title-main">Chorismate synthase</span>

The enzyme chorismate synthase catalyzes the chemical reaction

<span class="mw-page-title-main">Prephenate dehydratase</span>

The enzyme prephenate dehydratase (EC 4.2.1.51) catalyzes the chemical reaction

<span class="mw-page-title-main">Shikimate pathway</span> Biosynthetic Pathway

The shikimate pathway is a seven-step metabolic pathway used by bacteria, archaea, fungi, algae, some protozoans, and plants for the biosynthesis of folates and aromatic amino acids. This pathway is not found in animal cells.

<span class="mw-page-title-main">Hydrogen-bond catalysis</span>

Hydrogen-bond catalysis is a type of organocatalysis that relies on use of hydrogen bonding interactions to accelerate and control organic reactions. In biological systems, hydrogen bonding plays a key role in many enzymatic reactions, both in orienting the substrate molecules and lowering barriers to reaction. However, chemists have only recently attempted to harness the power of using hydrogen bonds to perform catalysis, and the field is relatively undeveloped compared to research in Lewis acid catalysis.

<span class="mw-page-title-main">Isochorismate lyase</span>

Isochorismate pyruvate lyase is an enzyme responsible for catalyzing part of the pathway involved in the formation of salicylic acid. More specifically, IPL will use isochorismate as a substrate and convert it into salicylate and pyruvate. IPL is a PchB enzyme originating from the pchB gene in Pseudomonas aeruginosa.

References

  1. 1 2 3 4 Qamra R, Prakash P, Aruna B, Hasnain SE, Mande SC (June 2006). "The 2.15 A crystal structure of Mycobacterium tuberculosis chorismate mutase reveals an unexpected gene duplication and suggests a role in host-pathogen interactions". Biochemistry. 45 (23): 6997–7005. doi:10.1021/bi0606445. PMID   16752890.
  2. 1 2 3 Kast P, Grisostomi C, Chen IA, Li S, Krengel U, Xue Y, Hilvert D (November 2000). "A strategically positioned cation is crucial for efficient catalysis by chorismate mutase". The Journal of Biological Chemistry. 275 (47): 36832–8. doi: 10.1074/jbc.M006351200 . PMID   10960481.
  3. ↑ Luk LY, Qian Q, Tanner ME (August 2011). "A cope rearrangement in the reaction catalyzed by dimethylallyltryptophan synthase?". Journal of the American Chemical Society. 133 (32): 12342–5. doi:10.1021/ja2034969. PMID   21766851.
  4. ↑ Selwood T, Jaffe EK (March 2012). "Dynamic dissociating homo-oligomers and the control of protein function". Archives of Biochemistry and Biophysics. 519 (2): 131–43. doi:10.1016/j.abb.2011.11.020. PMC   3298769 . PMID   22182754.
  5. ↑ Babu M (1999). "Annotation of Chorismate Mutase from the Mycobacterium tuberculosis and the Mycobacterium leprae genome" (PDF). Undergraduate Thesis for the Center of Biotechnology.
  6. 1 2 Lee AY, Stewart JD, Clardy J, Ganem B (April 1995). "New insight into the catalytic mechanism of chorismate mutases from structural studies". Chemistry & Biology. 2 (4): 195–203. doi: 10.1016/1074-5521(95)90269-4 . PMID   9383421.
  7. ↑ Gray JV, Knowles JR (August 1994). "Monofunctional chorismate mutase from Bacillus subtilis: FTIR studies and the mechanism of action of the enzyme". Biochemistry. 33 (33): 9953–9. doi:10.1021/bi00199a018. PMID   8061004.
  8. ↑ Grisham C (2017). Biochemistry 6th Edition. United States of America: Brooks/Cole - Cengage Learning. p. 505. ISBN   978-1133106296.
  9. ↑ Kienhöfer A, Kast P, Hilvert D (March 2003). "Selective stabilization of the chorismate mutase transition state by a positively charged hydrogen bond donor" . Journal of the American Chemical Society. 125 (11): 3206–7. doi:10.1021/ja0341992. PMID   12630863.
  10. ↑ Hur S, Bruice TC (October 2003). "The near attack conformation approach to the study of the chorismate to prephenate reaction". Proceedings of the National Academy of Sciences of the United States of America. 100 (21): 12015–20. doi: 10.1073/pnas.1534873100 . PMC   218705 . PMID   14523243.
  11. ↑ Strajbl M, Shurki A, Kato M, Warshel A (August 2003). "Apparent NAC effect in chorismate mutase reflects electrostatic transition state stabilization". Journal of the American Chemical Society. 125 (34): 10228–37. doi:10.1021/ja0356481. PMID   12926945.
  12. ↑ Burschowsky D, van Eerde A, Ökvist M, Kienhöfer A, Kast P, Hilvert D, Krengel U (December 2014). "Electrostatic transition state stabilization rather than reactant destabilization provides the chemical basis for efficient chorismate mutase catalysis". Proceedings of the National Academy of Sciences of the United States of America. 111 (49): 17516–21. Bibcode:2014PNAS..11117516B. doi: 10.1073/pnas.1408512111 . PMC   4267393 . PMID   25422475.
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