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Definition of Enzymes

Enzymes are biocatalysts, which accelerate chemical reactions and thus play a vital role in human metabolism. Almost always, they consist of proteins with a molecular weight in the range of about 10,000–100,000 Daltons. They are synthesized in the cell as part of protein biosynthesis.

An exception, however, is catalytically active nucleic acids, the so-called ribozymes such as the snRNA. The substances that are converted by enzymes are referred to as substrates, and the end result of enzymatic action is products.

Note: Most enzymes have ‘ase’ at the end of their name.


Enzymes as Biocatalysts

An enzyme will only catalyze a specific type of reaction using specific substrates.

Biocatalysts do not change the reaction pathway of a chemical reaction. Instead, they accelerate the reaction by lowering the activation energy. During the conversion of substrates (S) to products (P), the enzyme (E) is not consumed and exists unchanged after the reaction. The reaction pathway can be represented simply as follows:

E + S ES E + P

During the conversion of substrates to products, a transition state must be overcome which is unfavorable compared with the energetic state of substrate and product, indicating that it has higher free energy. The activation energy is thus the difference between the free energy of substrate and transition states.

In order to reduce the activation energy, enzymes interact with substrates, forming Enzyme-Substrate Complexes (ES). This stabilizes the transition state and its free energy is lowered. The binding of substrate occurs at the active center of the enzyme.

Activation Energy

Image: “Activation Energy” by Phil Schatz. License: CC BY 4.0

The Active Center

The active center is a three-dimensional, specifically folded region, comprising amino acids. It is usually arranged in a hollow shape. The conditions for chemical reactions are favored within this center, because the probability of overlapping reactants is improved by convergence and optimal orientation in space, among other things.

The binding of the substrate to the active center is not covalent. The substrate-binding occurs via:

  • Ionic interaction
  • Hydrophobic interaction
  • Van der Waals force
  • Hydrogen bridge bonds

The substrate-binding occurs according to the induced-fit model, which states that the active center changes its conformity after substrate binding such that it is complementary to the substrate. Previously, it was assumed that the conformation of the active center existed prior to substrate binding, which was referred to as the key-lock principle; this view is, however, outdated. 

Induced fit Model

Image: “ Induced fit Model” by Phil Schatz. License: CC BY 4.0

The 4 Enzyme Specificities

These specificities distinguish enzymes as biological catalysts from chemical catalysts (e.g., platinum). The 4 specificities of enzymes are:

1. Substrate Specificity

The induced-fit model forms the basis of the substrate specificity of enzymes. Accordingly, each enzyme can convert only one particular substrate; for example, in the context of glycolysis, hepatic glucokinase converts only glucose into glucose-6-phosphate and no other hexoses.

2. Stereospecificity

Also, the concept of stereoisomerism plays an important role: Lactate dehydrogenase, for example, can only convert L-lactate to pyruvate and not its mirror image D-lactate. The lactate dehydrogenase is thus stereospecific, or optically specific.

3. Group Specificity

If an enzyme reacts with a specific chemical group (e.g., amino group) that is located on the substrate, the substrate itself is not relevant, but rather the chemical group that it carries.

4. Reaction Specificity

The specificity of reaction states that each enzyme can catalyze only a specific type of reaction. For instance, the enzyme X catalyzes the reaction E + S ↔ ES ↔ E + P, but might not catalyze a similar reaction E + S ↔ ES ↔ E + P1 + P2.

Classification of Enzymes

Currently, there are more than 2,000 known enzymes that can be divided into 6 main groups, which, in turn, are categorized into different subclasses. The following table provides a good overview:

Main group Important subclasses + examples A brief explanation of the catalytic reaction
Oxidoreductases Dehydrogenases (alcohol dehydrogenase), oxidases (xanthine oxidase), reductases (glutathione reductase) Transfer of reduction equivalents (1 mole of electrons), in which the electron donor emits electrons and is oxidized, the electron acceptor receives the electrons and is reduced
Transferases Aminotransferases (ASAT/ aspartate aminotransferase), phosphotransferases (glycogen phosphorylase) Transfer of entire groups, e.g., amino groups
Hydrolases Esterases (acetylcholinesterase), peptidases/proteases (α-Amylase) Molecular fission with water addition = hydrolytic fission
Lyases C-C-Lyases (aldolase), C-O-Lyases (fumarase) Breaking of various chemical bonds by means other than hydrolysis, leaving a double bond. Independent of energy sources such as ATP
Isomerases cis-trans isomerases (PPI/ peptidyl-prolyl cis-trans isomerase) Conversion of isomeric molecules into each other without changing the molecular formula
Ligases C-C-ligases (pyruvate carboxylase), C-N-ligases (glutamine synthetase) Also called synthetases, energy-dependent linkage of compounds, i.e. dependent on substances such as ATP

Isoenzymes – Same Reaction, Different Structure

Some enzymes catalyze the same reaction, however, they differ slightly in their structure. Such enzymes are referred to as isoenzymes. Their amino acid sequence is different because they are coded by different genes.

They play an important role in medicine since different isoenzymes are typical for specific organs, and thus provide insights into pathological conditions. Increase in a specific isoenzyme in the blood represents a valuable diagnostic parameter for the detection of disease.

The best-known example is probably creatine kinase (CK). It consists of 2 subunits, the M-type (muscle) and B-type (brain), and has 3 isoforms. An increase of the CK-MB isoform, for example, can be traced to a heart attack since this enzyme is mainly localized in the heart muscle.

Another example is lactate dehydrogenase (LDH). It consists of four subunits, which are composed of the H-type (heart) and M-type (muscle). In the myocardium, the isoenzyme LDH1 exists consisting of 4 H subunits, whereas in the skeletal muscle, the isoenzyme LDH5 is present with 4 M subunits.

Coenzymes and Prosthetic Groups

However, enzymes are often dependent on auxiliary molecules, so-called coenzymes or co-substrates, without which a reaction might not be catalyzed. The auxiliary molecules can be firmly bound to the enzyme and are then called prosthetic groups, or exist in a soluble form.

They support the enzyme, for example, via transfer of electrons. The following table provides an overview of the most important coenzymes:

Coenzyme Function Example
NAD+/NADP+ Transfer of 2e and 1H+ Malate dehydrogenase
FAD Transfer of 2e und 2H+ Succinate dehydrogenase
Lipoamide Transfer of 2e und 2H+ Pyruvate dehydrogenase
Coenzyme Q / ubiquinone Transfer of 2e und 1H+ Respiratory chain
Cytochrome Transfer of 1e Respiratory chain
Biotin Transfer of carboxyl groups Pyruvate decarboxylase
Tetrahydrofolate (THF) Transfer of C1 groups Purine synthesis
Coenzyme A (CoA) Transfer of alkyl radicals α-ketoglutarate dehydrogenase
Thiamine pyrophosphate Transfer of hydroxyalkyl radicals Pyruvate dehydrogenase

Some enzymes, such as α-ketoglutarate dehydrogenase of the citric acid cycle, require several coenzymes; in this case, for example, thiamine pyrophosphate, lipoamide, FAD, and NAD+ in addition to CoA. 

Serine Proteases

  • Cleavage of peptide bonds
  • Specificity of cutting
  • Common active site composition/structure
  • Mechanistically well-studied

Kinetics of Enzymes

Enzyme kinetics describes the sequence of enzyme-catalyzed reactions depending on various parameters.

Dependence of reaction rate on temperature

One should remember the following thumb rule here: the reaction rate doubles for every 10° increase in temperature. This can certainly occur within a limited scope since enzymes exhibit an optimum temperature and denature at too high temperatures.

Dependence of the reaction rate on pH

There is no thumb rule as the one for temperature dependence. However, it is important to know that enzymes have a pH optimum and changes in the pH value alter functional groups of the enzyme or its substrate.

Changes in functional groups lead to changes in spatial structure and the bond at the active center is improved or deteriorated. In the case of stronger pH changes, it may even result in denaturation of the enzyme. The gastric enzymes are unique in this regard: Pepsin, for example, works effectively at a pH of about 2, whereas other enzymes would long have denatured.

Dependence of the reaction rate on substrate concentration

1. Michaelis-Menten model

Michaelis Menten Curve

Image: “Michaelis-Menten-Diagramm” by Thomas Shafee. License: CC BY-SA 4.0

The Michaelis-Menten model is an established explanation for this relation. If one plots the reaction rate of the reaction E + S ↔ ES ↔ E + P against the substrate concentration on a graph, the result is a hyperbolic curve, which approaches the maximum velocity vmax.

A simple example can be used to understand the graph: if you have 5 enzymes and add 0 substrates, the reaction rate is zero since there is nothing to be converted. If you now gradually add more and more substrate, the reaction rate approaches the maximum velocity. If all 5 enzymes are occupied with substrates, it leads to substrate saturation and the maximum velocity is reached.

An important concept in this context is the Michaelis-Menten constant (KM), as it corresponds to the substrate concentration at half-maximal velocity (½ vmax). Accordingly, half of all enzymes are occupied with the substrate, i.e. they form part of an enzyme-substrate complex. The Michaelis-Menten constant thus indicates the affinity of an enzyme for its substrate and is characteristic for the particular enzyme-substrate complex.

Note: high KM value → low affinity; low KM value → high affinity

Explanation: If the KM value is low, the half-maximal velocity occurs, even when the substrate concentration is low and the enzyme has a strong affinity for the substrate. If the KM value is large, however, the half-maximal velocity will only be achieved if the range of substrates is greater, since the enzyme has less affinity for the substrate. Therefore, the smaller the KM, the stronger is the reaction rate increase with the substrate concentration.

The Michaelis-Menten constant depends on temperature and pH value but is independent of the enzyme concentration.

To calculate the reaction rate of the enzyme-catalyzed reactions depending on the substrate concentration, the Michaelis-Menten equation is used. It states:

Michaelis Menten Gleichung

There are 3 possibilities:

  1. The substrate concentration S is significantly lower than KM. In this case, the addition of S to KM is negligible as it does not significantly increase the value of the denominator, and the equation is changed to v = vmax [S]/KM
  2. The substrate concentration is equal to KM. In this case, the denominator KM+ [S] can be changed to 2 [S]; the equation now reads v = vmax · [S]/2[S] and this can be shortened to v = ½ vmax
  3. The substrate concentration is significantly higher than KM. In this case, one can neglect KM during the addition in the denominator, so that the equation is v = vmax · [S]/[S] and the fraction can be shortened to v = vmax. This is due to substrate saturation under very high substrate concentrations, resulting in maximum velocity.

2. Lineweaver-Burk graph

Lineweaver Burk Diagram

Image: “ Lineweaver Burk Diagram” von Pro bug catcher. License: CC BY-SA 3.0

Also, the following graph is important. It is merely a different representation of the reaction rate, depending on substrate concentration. Based on a complex transformation of the Michaelis-Menten equation, a typical linear equation of the form y = mx + n is derived as follows:

linear equation

Note: Using the Lineweaver-Burk graph, one can directly read both KM as well as vmax. Thereby, -1/KM correspond to the intersection with the abscissa and 1/vmax to the intersection with the ordinate.

Regulation of Enzymes

The human body has the ability to stimulate or to curb metabolic processes via long- or short-term regulation of enzymes.

Short-term regulatory mechanisms include:

  • Competitive inhibition
  • Non-competitive inhibition
  • Uncompetitive inhibition
  • Interconversion
  • Allosteric regulation

The long-term regulatory mechanisms, in turn, include:

  • Increased synthesis of necessary enzymes
  • Increased reduction of unnecessary enzymes

Inhibition of Enzymes

Enzyme Inhibition

Bild: “Enzyme Inhibition” by Phil Schatz. License: CC BY 4.0

Three types of enzyme inhibition

In competitive inhibition, the inhibitor resembles the substrate and thus competes with it for the binding site at the active center of the enzyme. The inhibitor binds at the active center, thereby blocking this binding site. The KM value increases since an increased substrate concentration is required in order to obtain the half-maximal velocity.

However, Vmax is unchanged. The inhibitor can be removed from the bond under substrate excess; therefore, this inhibition is reversible. Examples: ACE inhibitors, Curare.

In non-competitive inhibition, the inhibitor does not have a similar substrate because it binds and inhibits the enzyme outside of the active center. The substrate can thus continue to bind to the active center but is not converted due to the additional binding of the inhibitor, implying that the number of functional enzyme-substrate complexes decreases.

This decreases Vmax while KM remains the same. A substrate excess does not displace the inhibitor. The non-competitive inhibition can be reversible or irreversible. Example: acetylsalicylic acid (irreversible).

Uncompetitive inhibition is a rare form of enzyme inhibition and characterized by specific binding at the enzyme-substrate complex. The inhibitor also binds outside the active center, but only if the enzyme-substrate complex is already formed. The result is a reversible conformational change, and thus inactivation of the enzyme. KM and vmax are reduced.

Irreversible Enzyme Inhibition: Suicide – Enzyme Inhibitors


Allosteric Effects

Allosteric Effects

Image: “Allosteric Effects” by Phil Schatz. License: CC BY 4.0

Allosteric regulation is the most common form of regulation and is facilitated by allosteric ligand/effectors binding outside the active center of the enzyme, and occurs at the allosteric center leading to a conformational change and thus, activation or deactivation of the enzyme.

In allosteric enzymes, the 2 states can be distinguished: the inactive T-form (tensed) and the active R-form (relaxed). Allosteric inhibitors thus stabilize the T-form and allosteric activators stabilize the R-form. Often, the substrate itself represents an allosteric activator, which makes sense because the substrate thereby promotes the R-condition for its own implementation.

Depending on the nature of allosteric effector, the maximum velocity, or the KM value, is changed. Effectors of the v-type increase/decrease the maximum velocity, and effectors from the k-type influence the KM value. However, the dependence of the reaction rate on substrate concentration in allosteric enzymes is not hyperbolic, but rather sigmoid.

The sigmoid relationship is due to the composition of the allosteric enzymes, usually consisting of several subunits and hence have more active centers, such that the binding of the first substrate, which at the same time is also the allosteric activator, facilitates the binding of the second substrate. This effect is called positive cooperativity.

However, if the binding of the first substrate complicates the binding of the second substrate, it is termed as negative cooperativity. Cooperativity occurs not only in enzymes but also in other proteins, e.g., hemoglobin.


Enzymes can also be regulated by interconversion, for example, specific groups (e.g., a phosphate residue) are added to, or cleaved off, of the enzyme, which changes the active state of the enzyme. Enzyme phosphorylation is mediated by kinases and dephosphorylation by phosphatases.

Enzyme Defects and their Impact


Enzymes can be limited in their activity, or fail completely, for e.g., due to genetic defects leading to an enzyme deficiency as in phenylketonuria (PKU). In this disease, the enzyme phenylalanine hydroxylase is defective, so that phenylalanine cannot be converted into tyrosine.

The accumulation of phenylalanine leads to the formation of neurotoxic substances, which, if left untreated, results in mental retardation with increased convulsions beginning in the 4th month of life. Treatment of choice is a controlled diet with reduced phenylalanine content. If this is already initiated in infancy, mental retardation can be prevented completely.

Myasthenia Gravis

Patient with Myasthenia Gravis

Image: “Patient with Myasthenia Gravis” by Phil Schatz. License: Public Domain

Sometimes, it is necessary to actually restrict the enzymatic activity, for e.g., in the case of myasthenia gravis disease, which manifests as increased muscle fatigue.

The disease is caused by antibodies targeting and blocking acetylcholine receptors of the muscle endplate. Treatment, therefore, consists of administration of acetylcholinesterase inhibitors such as pyridostigmine to reduce the degradation of the transmitter acetylcholine in the synaptic cleft, and maintain higher concentrations at the muscle endplate in order to trigger endplate potentials.

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