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:
There is only one substrate yielding one product. This product has the same molecular formula as the substrate but differs in bond connectivity or spatial arrangement. Isomerases catalyze reactions across many biological processes, such as in glycolysis and carbohydrate metabolism.
Isomerization
Isomerases catalyze changes within one molecule. They convert one isomer to another, meaning that the end product has the same molecular formula but a different physical structure. Isomers themselves exist in many varieties but can generally be classified as structural isomers or stereoisomers. Structural isomers have a different ordering of bonds and/or different bond connectivity from one another, as in the case of hexane and its four other isomeric forms (2-methylpentane, 3-methylpentane, 2,2-dimethylbutane, and 2,3-dimethylbutane).
Stereoisomers have the same ordering of individual bonds and the same connectivity but the three-dimensional arrangement of bonded atoms differ. For example, 2-butene exists in two isomeric forms: cis-2-butene and trans-2-butene. The sub-categories of isomerases containing racemases, epimerases and cis-trans isomers are examples of enzymes catalyzing the interconversion of stereoisomers. Intramolecular lyases, oxidoreductases and transferases catalyze the interconversion of structural isomers.
The prevalence of each isomer in nature depends in part on the isomerization energy, the difference in energy between isomers. Isomers close in energy can interconvert easily and are often seen in comparable proportions. The isomerization energy, for example, for converting from a stable cis isomer to the less stable trans isomer is greater than for the reverse reaction, explaining why in the absence of isomerases or an outside energy source such as ultraviolet radiation a given cis isomer tends to be present in greater amounts than the trans isomer. Isomerases can increase the reaction rate by lowering the isomerization energy.
Calculating isomerase kinetics from experimental data can be more difficult than for other enzymes because the use of product inhibition experiments is impractical. That is, isomerization is not an irreversible reaction since a reaction vessel will contain one substrate and one product so the typical simplified model for calculating reaction kinetics does not hold. There are also practical difficulties in determining the rate-determining step at high concentrations in a single isomerization. Instead, tracer perturbation can overcome these technical difficulties if there are two forms of the unbound enzyme. This technique uses isotope exchange to measure indirectly the interconversion of the free enzyme between its two forms. The radiolabeled substrate and product diffuse in a time-dependent manner. When the system reaches equilibrium the addition of unlabeled substrate perturbs or unbalances it. As equilibrium is established again, the radiolabeled substrate and product are tracked to determine energetic information.
The earliest use of this technique elucidated the kinetics and mechanism underlying the action of phosphoglucomutase, favoring the model of indirect transfer of phosphate with one intermediate and the direct transfer of glucose. This technique was then adopted to study the profile of proline racemase and its two states: the form which isomerizes L-proline and the other for D-proline. At high concentrations it was shown that the transition state in this interconversion is rate-limiting and that these enzyme forms may differ just in the protonation at the acidic and basic groups of the active site.
Nomenclature Generally, "the names of isomerases are formed as "substrate isomerase" (for example, enoyl CoA isomerase), or as "substrate type of isomerase" (for example, phosphoglucomutase)."
Classification Enzyme-catalyzed reactions each have a uniquely assigned classification number. Isomerase-catalyzed reactions have their own EC category: EC 5. Isomerases are further classified into six subclasses:
Racemases, epimerases This category (EC 5.1) includes (racemases) and epimerases). These isomerases invert stereochemistry at the target chiral carbon. Racemases act upon molecules with one chiral carbon for inversion of stereochemistry, whereas epimerases target molecules with multiple chiral carbons and act upon one of them. A molecule with only one chiral carbon has two enantiomeric forms, such as serine having the isoforms D-serine and L-serine differing only in the absolute configuration about the chiral carbon. A molecule with multiple chiral carbons has two forms at each chiral carbon. Isomerization at one chiral carbon of several yields epimers, which differ from one another in absolute configuration at just one chiral carbon. For example, D-glucose and D-mannose differ in configuration at just one chiral carbon. This class is further broken down by the group the enzyme acts upon:
+ Racemases and epimerases: EC number Description Examples EC 5.1.1 Acting on Amino Acids and Derivative alanine racemase, methionine racemase EC 5.1.2 Acting on Hydroxy Acids and Derivatives lactate racemase, tartrate epimerase EC 5.1.3 Acting on Carbohydrates and Derivatives ribulose-phosphate 3-epimerase, UDP-glucose 4-epimerase EC 5.1.99 Acting on Other Compounds methylmalonyl CoA epimerase, hydantoin racemase
Cis-trans isomerases This category (EC 5.2) includes enzymes that catalyze the isomerization of cis-trans isomers. Alkenes and cycloalkanes may have cis-trans stereoisomers. These isomers are not distinguished by absolute configuration but rather by the position of substituent groups relative to a plane of reference, as across a double bond or relative to a ring structure. Cis isomers have substituent groups on the same side and trans isomers have groups on opposite sides.
This category is not broken down any further. All entries presently include:
+ Cis-trans isomerases: EC number Examples EC 5.2.1.1 Maleate isomerase EC 5.2.1.2 Maleylacetoacetate isomerase EC 5.2.1.4 Maleylpyruvate isomerase EC 5.2.1.5 Linoleate isomerase EC 5.2.1.6 Furylfuramide isomerase EC 5.2.1.8 Peptidylprolyl isomerase EC 5.2.1.9 Farnesol 2-isomerase EC 5.2.1.10 2-chloro-4-carboxymethylenebut-2-en-1,4-olide isomerase EC 5.2.1.12 Zeta-carotene isomerase EC 5.2.1.13 Prolycopene isomerase EC 5.2.1.14 Beta-carotene isomerase
Intramolecular oxidoreductases This category (EC 5.3) includes intramolecular oxidoreductases. These isomerases catalyze the transfer of electrons from one part of the molecule to another. In other words, they catalyze the oxidation of one part of the molecule and the concurrent reduction of another part. Sub-categories of this class are:
+ Intramolecular oxidoreductases: EC number Description Examples EC 5.3.1 Interconverting Aldoses and Ketoses Triose-phosphate isomerase, Ribose-5-phosphate isomerase EC 5.3.2 Interconverting Keto- and Enol-Groups Phenylpyruvate tautomerase, Oxaloacetate tautomerase EC 5.3.3 Transposing C=C Double Bonds Steroid Delta-isomerase, L-dopachrome isomerase EC 5.3.4 Transposing S-S Bonds Protein disulfide-isomerase EC 5.3.99 Other Intramolecular Oxidoreductases Prostaglandin-D synthase, Allene-oxide cyclase
Intramolecular transferases This category (EC 5.4) includes intramolecular transferases (mutases). These isomerases catalyze the transfer of functional groups from one part of a molecule to another. Phosphotransferases (EC 5.4.2) were categorized as transferases (EC 2.7.5) with regeneration of donors until 1983. This sub-class can be broken down according to the functional group the enzyme transfers:
+ Intramolecular transferases: EC number Description Examples EC 5.4.1 Transferring Acyl Groups Lysolecithin acylmutase, Precorrin-8X methylmutase EC 5.4.2 Phosphotransferases (Phosphomutases) Phosphoglucomutase, Phosphopentomutase EC 5.4.3 Transferring Amino Groups Beta-lysine 5,6-aminomutase, Tyrosine 2,3-aminomutase EC 5.4.4 Transferring hydroxy groups (hydroxyamino)benzene mutase, Isochorismate synthase EC 5.4.99 Transferring Other Groups Methylaspartate mutase, Chorismate mutase
Intramolecular lyases This category (EC 5.5) includes intramolecular lyases. These enzymes catalyze "reactions in which a group can be regarded as eliminated from one part of a molecule, leaving a double bond, while remaining covalently attached to the molecule." Some of these catalyzed reactions involve the breaking of a ring structure.
This category is not broken down any further. All entries presently include:
+ Intramolecular lyases: EC number Examples EC 5.5.1.1 Muconate cycloisomerase EC 5.5.1.2 3-carboxy-cis,cis-muconate cycloisomerase EC 5.5.1.3 Tetrahydroxypteridine cycloisomerase EC 5.5.1.4 Inositol-3-phosphate synthase EC 5.5.1.5 Carboxy-cis,cis-muconate cyclase EC 5.5.1.6 Chalcone isomerase EC 5.5.1.7 Chloromuconate cycloisomerase EC 5.5.1.8 (+)-bornyl diphosphate synthase EC 5.5.1.9 Cycloeucalenol cycloisomerase EC 5.5.1.10 Alpha-pinene-oxide decyclase EC 5.5.1.11 Dichloromuconate cycloisomerase EC 5.5.1.12 Copalyl diphosphate synthase EC 5.5.1.13 Ent-copalyl diphosphate synthase EC 5.5.1.14 Syn-copalyl-diphosphate synthase EC 5.5.1.15 Terpentedienyl-diphosphate synthase EC 5.5.1.16 Halimadienyl-diphosphate synthase EC 5.5.1.17 (S)-beta-macrocarpene synthase EC 5.5.1.18 Lycopene epsilon-cyclase EC 5.5.1.19 Lycopene beta-cyclase EC 5.5.1.20 Prosolanapyrone-III cycloisomerase EC 5.5.1.n1 D-ribose pyranase
Mechanisms of isomerases
Ring expansion and contraction via tautomers
A classic example of ring opening and contraction is the isomerization of glucose (an aldehyde with a six-membered ring) to fructose (a ketone with a five-membered ring). The conversion of D-glucose-6-phosphate to D-fructose-6-phosphate is catalyzed by glucose-6-phosphate isomerase, an intramolecular oxidoreductase. The overall reaction involves the opening of the ring to form an aldose via acid/base catalysis and the subsequent formation of a cis-endiol intermediate. A ketose is then formed and the ring is closed again.
Glucose-6-phosphate first binds to the active site of the isomerase. The isomerase opens the ring: its His388 residue protonates the oxygen on the glucose ring (and thereby breaking the O5-C1 bond) in conjunction with Lys518 deprotonating the C1 hydroxyl oxygen. The ring opens to form a straight-chain aldose with an acidic C2 proton. The C3-C4 bond rotates and Glu357 (assisted by His388) depronates C2 to form a double bond between C1 and C2. A cis-endiol intermediate is created and the C1 oxygen is protonated by the catalytic residue, accompanied by the deprotonation of the endiol C2 oxygen. The straight-chain ketose is formed. To close the fructose ring, the reverse of ring opening occurs and the ketose is protonated.
Epimerization
An example of epimerization is found in the Calvin cycle when D-ribulose-5-phosphate is converted into D-xylulose-5-phosphate by ribulose-phosphate 3-epimerase. The substrate and product differ only in stereochemistry at the third carbon in the chain. The underlying mechanism involves the deprotonation of that third carbon to form a reactive enolate intermediate. The enzyme's active site contains two Asp residues. After the substrate binds to the enzyme, the first Asp deprotonates the third carbon from one side of the molecule. This leaves a planar sp2-hybridized intermediate. The second Asp is located on the opposite side of the active side and it protonates the molecule, effectively adding a proton from the back side. These coupled steps invert stereochemistry at the third carbon.
Intramolecular transfer