The addition and elimination of water are common modes of chemical transformation in metabolic networks. Unlike hydrolysis reactions, which use water to directly cleave chemical bonds, hydro-lyases generally form double bonds through dehydration or alter the positions of double bonds and hydroxyl groups through hydration, thereby completing structural rearrangement of substrates.

Keywords: hydro-lyases; fumarate hydratase; aconitate hydratase; enolase; enoyl-CoA hydratase; carbonic anhydrase

1 Reaction Basis of Hydro-Lyases

1.1 Hydration and Dehydration Reactions

Typical reactions catalyzed by hydro-lyases involve elimination of H and OH from adjacent atoms to form a double bond, or, in the reverse reaction, addition of H and OH across a double bond. Their reaction chemistry is fundamentally different from that of hydrolases, which use water to directly cleave ester, amide, or glycosidic bonds. Many hydro-lyase reactions are reversible, so the same enzyme may be named Hydratase or Dehydratase according to the predominant physiological reaction direction. For example, Fumarate Hydratase is usually described in the direction of Fumarate hydration to form L-Malate, whereas Enolase is generally described in the direction of 2-Phosphoglycerate dehydration to form Phosphoenolpyruvate.

1.2 Diversity of Catalytic Centers

(1) Acid-Base Catalysis and Substrate Positioning

Some hydro-lyases mainly rely on active-site acid-base groups and precise substrate positioning to control reaction direction. Fumarate Hydratase, for example, restricts the spatial orientation of Fumarate within the catalytic pocket, allowing water to add across the C=C double bond in a defined direction and thereby producing L-Malate with a specific stereochemical configuration.

(2) Fe-S Cluster-Dependent Systems

Aconitase and some metabolic dehydratases depend on Fe-S clusters for catalysis. In Aconitase, the [4Fe-4S] cluster is not merely a structural component; one iron atom directly participates in substrate coordination, allowing Citrate and cis-Aconitate to adopt suitable conformations for sequential dehydration and rehydration.

(3) Metal Ion-Dependent Systems

Enolase requires Mg²⁺ for substrate binding and stabilization of negatively charged states formed during the reaction, whereas mammalian α-type Carbonic Anhydrase depends on Zn²⁺ to lower the effective pKa of coordinated water and generate nucleophilic OH⁻. Therefore, hydro-lyases do not share a uniform metal-dependence pattern, and their redox sensitivity, metal requirements, and inhibition mechanisms need to be evaluated according to the specific enzyme.

1.3 Chemical Basis of Substrate Selectivity

(1) Recognition of Positioning Groups

Hydro-lyase reactions require precise control over the positions at which H and OH are eliminated or added, so the active site usually recognizes multiple substrate structural features simultaneously. Carboxyl groups, phosphate groups, CoA thioesters, and hydrophobic acyl chains not only determine binding strength but also contribute to substrate orientation, positioning the reaction center appropriately in space.

(2) Regioselectivity and Stereoselectivity

After the substrate is fixed in place, catalytic groups further restrict the direction of proton transfer and water attack, causing double-bond formation or hydration to occur at specific carbon atoms and controlling the configuration of newly formed chiral centers. Regioselectivity and stereoselectivity therefore constitute the core chemical basis of substrate specificity in hydro-lyases.

Table 1 Catalytic Characteristics of Representative Hydro-Lyases

2 Fumarate Hydratase

2.1 Reversible Conversion Between Fumarate and L-Malate

Fumarate Hydratase, also known as Fumarase, catalyzes hydration of the Fumarate double bond to form L-Malate and, in the reverse direction, catalyzes dehydration of L-Malate to form Fumarate. This reaction does not alter carbon-skeleton length and involves no net electron transfer. Its core chemistry is the addition of H and OH from water across the C=C double bond of Fumarate in a defined spatial orientation. The enzyme active site imposes strict restrictions on substrate orientation, so the reaction produces L-Malate with a specific configuration rather than a random mixture of stereoisomers.

2.2 Functional Positioning in the TCA Cycle

In the mitochondrial TCA cycle, Succinate is oxidized by Succinate Dehydrogenase to form Fumarate, which is then converted by Fumarate Hydratase into L-Malate and subsequently oxidized by Malate Dehydrogenase to form Oxaloacetate. This step does not directly generate NADH, FADH₂, or ATP, but it determines whether the Fumarate carbon skeleton can continue to be transferred toward Oxaloacetate. Therefore, reduced FH function can simultaneously affect the Fumarate/Malate balance, continuity of the TCA cycle, and related carbon-flux distribution.

2.3 FH Deficiency and Fumarate Accumulation

(1) Protein Succination

FH dysfunction can lead to marked accumulation of Fumarate. Fumarate has a certain degree of electrophilicity and can undergo nonenzymatic addition with protein Cysteine thiol groups to form S-(2-succino)cysteine, or 2SC. This process is known as Succination and is chemically distinct from lysine Succinylation involving Succinyl-CoA. Increased 2SC under FH-deficient conditions can reflect sustained Fumarate exposure and may alter the structure or function of key proteins.

(2) α-Ketoglutarate-Dependent Reactions

High levels of Fumarate can also interfere with some α-Ketoglutarate-dependent dioxygenases, extending the effects of FH abnormalities from central carbon metabolism to regulatory processes such as oxygen sensing, DNA modification, and histone modification. Therefore, the biological effects of FH deficiency arise not only from insufficient L-Malate formation but also from abnormal accumulation of upstream Fumarate.

2.4 Interpretation of the Fumarate/Malate Relationship

(1) Metabolite Changes

Increased Fumarate accompanied by a relative decrease in Malate can serve as an important metabolic feature indicating restriction at the FH reaction node. However, Fumarate levels are also affected by Succinate oxidation, bypass metabolism, and cellular compartmentalization, whereas Malate additionally participates in the malate-aspartate shuttle, gluconeogenesis, and other anaplerotic pathways.

(2) Enzyme Activity Evaluation

The Fumarate/Malate ratio is suitable for evaluating the state of the reaction node but cannot directly replace FH enzyme activity. When FH catalytic function itself needs to be assessed, metabolite measurements should be combined with direct enzyme-activity analysis, protein-level detection, or isotope-tracing results.

3 Aconitate Hydratase/Aconitase

3.1 Two-Step Isomerization Between Citrate and Isocitrate

(1) Citrate Dehydration

Aconitase does not directly move the hydroxyl group in Citrate to another position. Instead, it first removes H and OH in a direction defined by the active site, forming cis-Aconitate containing a C=C double bond. This step requires the enzyme to distinguish among carbon atoms and carboxyl groups in Citrate that have similar chemical environments so that dehydration occurs at a defined position.

(2) Rehydration of cis-Aconitate

cis-Aconitate subsequently binds water again, but the positions at which H and OH are added differ from those in the original formation of Citrate, ultimately producing Isocitrate. The sequential “dehydration-rehydration” process rearranges the hydroxyl-group position and creates the appropriate structure for the subsequent oxidative decarboxylation catalyzed by Isocitrate Dehydrogenase.

3.2 Catalytic Role of the [4Fe-4S] Cluster

The Aconitase active site contains a [4Fe-4S] cluster, one iron atom of which directly participates in coordination of Citrate, cis-Aconitate, or Isocitrate. This Fe-S cluster differs from iron-sulfur centers that mainly function in electron transfer. Its primary role is to organize substrate-binding conformation and participate in hydration/dehydration catalysis. Therefore, the integrity of the Fe-S cluster directly determines whether Aconitase can maintain normal catalytic activity.

3.3 Mitochondrial Aconitase and the TCA Cycle

Mitochondrial ACO2 performs the major function of converting Citrate to Isocitrate in the TCA cycle. When ACO2 activity decreases, transfer of Citrate toward subsequent oxidative decarboxylation is restricted. However, Citrate can also be exported to the cytoplasm and participate in lipid synthesis, Acetyl-CoA supply, and acetylation reactions. Therefore, increased Citrate alone does not point to a single mechanism. The Aconitase node is more appropriately evaluated through integrated changes in Citrate, Isocitrate, and downstream α-Ketoglutarate.

3.4 ACO1/IRP1 and Iron Homeostasis

(1) Aconitase State

Cytoplasmic ACO1 is also known as IRP1. When its [4Fe-4S] cluster is intact, IRP1 mainly exists as cytoplasmic Aconitase and exhibits enzymatic catalytic activity, allowing the Fe-S cluster state to directly determine its functional state.

(2) IRE-Binding State

When the Fe-S cluster is lost, IRP1 undergoes a conformational change and acquires the ability to bind iron-responsive elements (IREs). By binding IREs in different mRNAs, it regulates translation or mRNA stability of proteins involved in iron uptake, storage, and utilization. This functional switch directly links cellular iron availability with gene-expression regulation.

3.5 Aconitase and Oxidative Stress

(1) Fe-S Cluster Damage

The Fe-S center of Aconitase is relatively sensitive to oxidative and nitrosative environments. Reactive oxygen species, NO, and related reactive species can damage or remodel the Fe-S cluster, thereby reducing Aconitase catalytic activity. In IRP1, such changes may also promote conversion of the protein to the IRE-binding state.

(2) Experimental Interpretation

A decrease in Aconitase activity detected under oxidative-stress conditions should not be directly interpreted as reduced protein expression. Functional damage to the Fe-S cluster can markedly reduce enzyme activity even when protein abundance changes little. Therefore, enzyme activity should be analyzed together with protein expression or indicators of Fe-S status.

4 Enolase

4.1 Dehydration of 2-Phosphoglycerate and Formation of PEP

(1) Formation of a Negatively Charged Intermediate State

Enolase catalyzes the dehydration of 2-Phosphoglycerate to form Phosphoenolpyruvate (PEP). During catalysis, a proton is first removed from a specific position, generating a negatively charged intermediate state that requires stabilization by metal ions.

(2) Formation of the Double Bond

Hydroxyl elimination then occurs and a C=C double bond is formed, generating PEP. This structure has high phosphoryl-transfer potential, allowing PEP to drive ADP phosphorylation and ATP production in the subsequent Pyruvate Kinase reaction.

4.2 Catalytic Role of Mg²⁺

(1) Substrate Binding

Mg²⁺ coordinates with oxygen-containing groups of 2-Phosphoglycerate, helping to define the substrate conformation within the active site and maintain an appropriate distance between the reacting carbon atoms and catalytic groups.

(2) Charge Stabilization

The negatively charged state formed after deprotonation of 2-Phosphoglycerate would be unfavorable for the subsequent elimination reaction without stabilization. Mg²⁺ lowers the energy of this intermediate state and is therefore part of the catalytic chemistry of Enolase rather than merely an auxiliary ion that maintains protein structural stability.

4.3 Functional Differentiation of Enolase Isoforms

(1) ENO1

ENO1, also known as α-Enolase, is widely expressed in many tissues and cells and is an important component of basal glycolysis. Changes in its expression or activity can affect the later stages of glycolysis, but overall glycolytic flux is simultaneously regulated by multiple nodes, including glucose uptake, Phosphofructokinase, Pyruvate Kinase, and mitochondrial pyruvate utilization. Therefore, ENO1 alone cannot serve as a surrogate for overall glycolytic intensity.

(2) ENO2

ENO2, also known as γ-Enolase or Neuron-Specific Enolase (NSE), is mainly associated with research on neurons and neuroendocrine-related cells. ENO2 retains full glycolytic catalytic function, so studies need to distinguish its role as a metabolic enzyme from its application as a biomarker associated with specific tissue origin or injury.

(3) ENO3

ENO3, also known as β-Enolase, is mainly associated with glucose metabolism in tissues such as skeletal muscle. ENO1, ENO2, and ENO3 catalyze the same type of core reaction, but the biological significance represented by changes in different isoforms depends on tissue origin, cell type, and metabolic background.

4.4 Enolase and Glycolytic Energy Conversion

Enolase itself does not directly generate ATP. Its key function is to use dehydration to restructure the electron distribution and bond-energy state of 2-Phosphoglycerate, generating PEP with high phosphoryl-transfer potential. PEP subsequently enters the Pyruvate Kinase reaction and drives substrate-level phosphorylation. Therefore, Enolase is located at an important transition point in glycolysis between structural rearrangement and direct ATP generation.

5 Enoyl-CoA Hydratase

5.1 Stereoselective Hydration of the Enoyl-CoA Double Bond

Enoyl-CoA Hydratase catalyzes the addition of water across the C2=C3 double bond of trans-2-Enoyl-CoA, with the classical reaction generating (3S)-3-Hydroxyacyl-CoA. The active site needs to recognize both the CoA moiety and the hydrophobic acyl chain and to control the direction from which H and OH are added to the double bond. Therefore, the β-hydroxyl group formed after hydration has a defined stereochemical configuration.

5.2 Role in Fatty Acid β-Oxidation

Fatty acid β-oxidation begins with formation of a trans double bond between the α and β carbons by Acyl-CoA Dehydrogenase. Enoyl-CoA Hydratase then hydrates this double bond to generate 3-Hydroxyacyl-CoA, which subsequently enters β-hydroxyl oxidation and thiolysis steps. This hydration reaction itself does not directly generate NADH or Acetyl-CoA but provides the hydroxyl structure required for subsequent β-position oxidation.

5.3 Substrate Range of ECHS1

(1) Fatty Acid Oxidation Substrates

Human ECHS1 is localized in the mitochondrial matrix and can catalyze hydration of multiple short-chain trans-2-Enoyl-CoA species, thereby participating in processing intermediates related to mitochondrial fatty acid β-oxidation.

(2) Branched-Chain Amino Acid Metabolism Substrates

ECHS1 can also act on unsaturated CoA esters such as Methacrylyl-CoA formed during Valine catabolism. Therefore, the physiological function of ECHS1 cannot be classified solely under classical fatty acid β-oxidation. Valine catabolism and processing of reactive unsaturated CoA intermediates are also important metabolic functions.

5.4 Accumulation of Reactive Enoyl-CoA Species

When ECHS1 function decreases, α,β-unsaturated CoA esters such as Methacrylyl-CoA and Acryloyl-CoA may accumulate abnormally. These intermediates are electrophilic and can undergo addition reactions with cellular nucleophilic groups such as Cysteine, forming abnormal metabolites or protein modifications. Therefore, part of the metabolic damage caused by ECHS1 deficiency results from failure to clear reactive upstream intermediates rather than simply from insufficient formation of hydration products.

5.5 Differences Among Enoyl-CoA Hydratase Systems

(1) Mitochondrial Hydration Systems

ECHS1 mainly carries out hydration of certain short-chain Enoyl-CoA species, whereas the hydration step in long-chain fatty acid β-oxidation can also be performed by mitochondrial multienzyme systems associated with long-chain fatty acid oxidation. Different systems have different substrate-chain-length preferences and protein compositions.

(2) Peroxisomal Hydration Systems

Peroxisomes also contain Enoyl-CoA Hydratase-related functions, with substrate ranges, subcellular localization, and metabolic backgrounds different from those of mitochondrial systems. Therefore, experimental studies should clearly define the specific protein, cellular compartment, and substrate type rather than using “Enoyl-CoA Hydratase” to summarize all reactions.

6 Carbonic Anhydrase

6.1 Rapid Reversible Conversion Between CO₂ and HCO₃⁻

Carbonic Anhydrase catalyzes the rapid reversible reaction of CO₂ hydration and HCO₃⁻ dehydration. CO₂ can undergo spontaneous hydration in water, but the rate is insufficient to meet the rapid gas-transport and acid-base-regulation demands of some tissues. Therefore, the core function of Carbonic Anhydrase is to accelerate equilibration of the CO₂/HCO₃⁻ system rather than unidirectionally increasing one particular product.

6.2 Zn²⁺-Activated Water and Proton Transfer

(1) CO₂ Hydration

Using mammalian α-type Carbonic Anhydrase II as an example, Zn²⁺ in the active site lowers the effective pKa of the coordinated water molecule, allowing formation of strongly nucleophilic Zn-bound OH⁻. This hydroxide attacks the carbon atom of CO₂ to form HCO₃⁻. HCO₃⁻ then leaves the active site and is replaced by a new water molecule.

(2) Proton Transfer

The newly bound Zn—H₂O must lose a proton to regenerate the Zn—OH⁻ state. In CA II, His64 and its connected water-molecule network participate in proton transfer from the metal center to the bulk buffer system. This process directly affects enzyme turnover rate, so the high catalytic efficiency of Carbonic Anhydrase depends on both Zn²⁺ activation and rapid proton transfer.

6.3 Spatial Division of Labor Among Carbonic Anhydrase Isoforms

(1) Cytoplasmic and Mitochondrial Isoforms

CA I and CA II are mainly located in the cytoplasm, whereas CA VA and CA VB are localized in mitochondria. Carbonic Anhydrases in different compartments can separately connect intracellular acid-base balance, CO₂ metabolism, and HCO₃⁻-dependent mitochondrial biosynthetic processes.

(2) Membrane-Associated and Secreted Isoforms

CA IV and related isoforms are located on the cell surface, CA VI is secreted, while CA IX and CA XII are transmembrane members. Differences in spatial localization allow different isoforms to couple with specific ion-transport systems and local pH environments. Therefore, mechanistic studies need to define the specific CA isoform rather than relying only on total CA activity.

6.4 Acid-Base Homeostasis and Inorganic Carbon Utilization

(1) Coupling of CO₂/HCO₃⁻ Transport

CO₂ can cross biological membranes relatively rapidly, whereas transmembrane HCO₃⁻ transport generally depends on specific transport proteins. By rapidly converting CO₂ and HCO₃⁻ on both sides of the membrane, Carbonic Anhydrase enables effective coupling between gas diffusion and ion transport.

(2) Local Substrate Supply

In mitochondria, kidneys, and other specific tissue environments, rapid CO₂/HCO₃⁻ conversion can also provide local substrates for HCO₃⁻-dependent metabolic reactions and acid-base regulation. Therefore, the physiological function of Carbonic Anhydrase is jointly determined by catalytic rate, spatial localization, and transport systems.

6.5 Mechanistic Basis of Sulfonamide Inhibitors

(1) Zn²⁺ Coordination Inhibition

Many classical Carbonic Anhydrase inhibitors contain sulfonamide structures. The deprotonated sulfonamide nitrogen can coordinate with Zn²⁺ in the active site and replace the position associated with the normal catalytic water, thereby reducing CO₂/HCO₃⁻ conversion capacity.

(2) Isoform Selectivity

Although different CA isoforms have similar metal catalytic cores, residues surrounding the active pocket differ. By altering substituents around the sulfonamide scaffold, different degrees of isoform selectivity can be achieved. Compounds such as Brinzolamide can be used for pharmacological inhibition of Carbonic Anhydrase and studies of isoform function.

7 Other Metabolism-Related Hydro-Lyases

7.1 Dihydroxy-Acid Dehydratase

Dihydroxy-acid Dehydratase catalyzes dehydration of dihydroxy acids such as 2,3-Dihydroxy-3-methylbutanoate to form the corresponding α-Keto Acid and is an important node in de novo biosynthesis of branched-chain amino acids such as Valine and Isoleucine. This pathway mainly exists in plants and microorganisms. These enzymes generally also show Fe-S center dependence and therefore represent important targets linking hydro-lyase chemistry, Fe-S enzymology, and branched-chain amino acid biosynthesis.

7.2 3-Dehydroquinate Dehydratase

3-Dehydroquinate Dehydratase catalyzes dehydration of 3-Dehydroquinate to form 3-Dehydroshikimate and is an important reaction in the shikimate pathway. Type I and Type II enzymes complete the same overall reaction but have different protein structures and catalytic mechanisms. Type I systems can form a Schiff-base intermediate, whereas Type II systems use a different acid-base catalytic strategy, representing distinct catalytic solutions formed through convergent evolution for the same hydro-lyase chemistry.

7.3 Imidazoleglycerol-Phosphate Dehydratase

Imidazoleglycerol-phosphate Dehydratase catalyzes dehydration of Imidazoleglycerol Phosphate to form Imidazole Acetol Phosphate during Histidine biosynthesis. This reaction mainly exists in plant and microbial systems capable of de novo Histidine synthesis, demonstrating that hydro-lyase reactions participate not only in energy and lipid metabolism but are also widely embedded in biosynthetic networks for amino acids and other fundamental metabolites.

8 Products Related to Hydro-Lyases and Metabolic Research

8.1 FH and Aconitase Reaction Systems

8.2 Enolase and Enoyl-CoA Hydratase Reaction Systems

8.3 Carbonic Anhydrase and Other Hydro-Lyase Reaction Systems

Overall, hydro-lyases share the chemical features of hydration or dehydration reactions, but their catalytic systems are not uniform. FH, Aconitase, Enolase, Enoyl-CoA Hydratase, and Carbonic Anhydrase use different mechanisms involving acid-base catalysis, Fe-S clusters, Mg²⁺, or Zn²⁺ to achieve substrate orientation and reaction control and connect the TCA cycle, glycolysis, fatty acid and branched-chain amino acid metabolism, and acid-base homeostasis.