酵素

酵素 (/ˈɛnzaɪmz/) は、化学反応を促進することによって生物学的触媒として働くタンパク質である。 酵素が作用する分子は基質と呼ばれ、酵素は基質を生成物として知られる異なる分子に変換する。細胞内のほとんどすべての代謝過程は、生命を維持するのに十分な速さで起こるために酵素触媒を必要とする。 代謝経路は、個々のステップを触媒する酵素に依存している。酵素の研究は酵素学と呼ばれ、擬似酵素分析の分野では、進化の過程で一部の酵素が生物学的触媒作用を遂行する能力を失っていることを認識している。このことはしばしばアミノ酸配列や特異な「擬触媒」特性に反映されている。

酵素グルコシダーゼは糖マルトースを2つのグルコースに変換する。赤が活性部位残基、黒がマルトース基質、NAD残基である。黄色は補因子。(PDB: 1OBB​)

酵素は5,000種類以上の生化学反応を触媒することが知られている。他の生体触媒は触媒RNA分子で、リボザイムと呼ばれる。酵素の特異性は、そのユニークな立体構造に由来する。

IUPACによる酵素の定義

すべての触媒と同様に、酵素はその活性化エネルギーを下げることによって反応速度を増加させる。一部の酵素は、基質から生成物への変換を何百万倍も速くすることができる。極端な例としては、オロチジン5'-リン酸脱炭酸酵素があり、この酵素を用いると、通常なら数百万年かかる反応が数ミリ秒で起こるようになる。化学的には、酵素は他の触媒と同様であり、化学反応で消費されることはなく、反応の平衡を変化させることもない。酵素は他のほとんどの触媒と異なり、はるかに特異的である。酵素活性は他の分子によって影響を受けることがある： 阻害剤は酵素活性を減少させる分子であり、活性化剤は活性を増加させる分子である。多くの治療薬物や毒物は酵素阻害剤である。酵素の活性は最適な温度とpH以外では著しく低下し、多くの酵素は過剰な熱にさらされると（永久的に）変性し、その構造と触媒特性を失う。

酵素の中には、例えば抗生物質の合成など、商業的に利用されているものもある。生物学的洗濯粉に含まれる酵素は衣服についたタンパク質やデンプン、脂肪の汚れを分解し、肉軟化剤に含まれる酵素はタンパク質をより小さな分子に分解して肉を噛み砕きやすくする。

語源と歴史

Eduard Buchner

17世紀後半から18世紀初頭までに、胃分泌物による肉の消化と、植物抽出物と唾液によるデンプンの糖への変換は知られていたが、これらが起こるメカニズムは特定されていなかった。

フランスの化学者Anselme Payenは1833年に初めて酵素であるジアスターゼを発見した。その数十年後、酵母による糖のアルコールへの発酵を研究していたルイ・パスツールは、この発酵は「発酵物」と呼ばれる酵母細胞内に含まれる生命力によって引き起こされると結論づけた。彼は「アルコール発酵は酵母細胞の生命と組織に関連した行為であり、細胞の死や腐敗に関連した行為ではない」と書いた。

1877年、ドイツの生理学者Wilhelm Kühne（1837-1900）が初めて酵素という用語を使用し、from Ancient Greek ἔνζυμον (énzymon) 'イーストで捏ねる'このプロセスを説明した。

1897年にエドゥアルド・ブフナーが酵母エキスの研究に関する最初の論文を提出した。彼はベルリン大学での一連の実験において、混合物中に生きた酵母細胞が存在しなくても、酵母エキスによって砂糖が発酵することを発見した。彼はショ糖の発酵をもたらす酵素を「ザイマーゼ」と名付けた。1907年、彼は「無細胞発酵の発見」によりノーベル化学賞を受賞した。ブフナーの例に倣い、酵素は通常、それらが行う反応に従って命名される。接尾辞-aseは基質の名前と組み合わされる（例えば、ラクターゼは乳糖を切断する酵素である）、あるいは反応の種類に組み合わされる（例えば、DNAポリメラーゼはDNAポリマーを形成する）。

酵素の生化学的な正体は、1900年代初頭にはまだ不明であった。多くの科学者は酵素活性がタンパク質と関連していることを観察していたが、他の科学者（ノーベル賞受賞者のリチャード・ウィルシュテッターなど）は、タンパク質は真の酵素の単なるキャリアであり、タンパク質それ自体は触媒作用ができないと主張していた。1926年、ジェームズ・B・サムナーはウレアーゼという酵素が純粋なタンパク質であることを示し、それを結晶化させた。1937年にはカタラーゼという酵素も同様に結晶化させた。純粋なタンパク質が酵素になりうるという結論は、消化酵素ペプシン（1930年）、トリプシン、キモトリプシンの研究を行ったジョン・ハワード・ノースロップとウェンデル・メレディス・スタンリーによって決定的に示された。これら3人の科学者は1946年にノーベル化学賞を受賞した。

酵素が結晶化できることが発見されると、最終的にはX線結晶構造解析によってその構造を解明することができるようになった。これが最初に行われたのはリゾチームで、涙や唾液、卵白に含まれ、いくつかのバクテリアの被膜を消化する酵素である。その構造はデイヴィッド・チルトン・フィリップスが率いるグループによって解明され、1965年に発表された。このリゾチームの高分解能構造は、構造生物学の分野の始まりであり、酵素がどのように働くのかを原子レベルの詳細さで理解しようとする努力の始まりであった。

分類と命名法

酵素は主に2つの基準で分類することができる：アミノ酸配列の類似性（したがって進化的関係）と酵素活性である。

酵素活性。酵素の名前は多くの場合、その基質または触媒する化学反応に由来し、語尾に-aseが付く。例えば、ラクターゼ、アルコールデヒドロゲナーゼ、DNAポリメラーゼなどである。同じ化学反応を触媒する異なる酵素はアイソザイムと呼ばれる。

国際生化学分子生物学連合は、酵素の命名法であるEC番号（「酵素委員会」の意）を開発した。 各酵素は "EC "の後に、酵素活性の階層（非常に一般的なものから非常に特異的なものまで）を表す4つの番号のシーケンスで記述される。つまり、最初の数字はそのメカニズムに基づいて酵素を大まかに分類し、他の数字はより特異性を高めていく。

トップレベルの分類は以下の通りである：

• EC 1の酸化還元酵素：酸化/還元反応を触媒する。
• EC 2, 転移酵素：官能基（メチル基やリン酸基など）を転移する。
• EC 3, ヒドロラーゼ：様々な結合の加水分解を触媒する
• EC 4, リアーゼ：加水分解と酸化以外の方法で様々な結合を切断する
• EC 5, イソメラーゼ：1分子内の異性体化変化を触媒する
• EC 6, リガーゼ：2つの分子を共有結合で結合させる。
• EC 7, トランスロカーゼ：膜を介したイオンや分子の移動、または膜内での分離を触媒する。

これらのセクションは、基質、生成物、化学機構などの他の特徴によって細分化される。酵素は4つの数値で完全に指定される。例えば、ヘキソキナーゼ（EC 2.7.1.1）は、アルコール基（EC 2.7.1）を含む分子であるヘキソース糖にリン酸基（EC 2.7）を付加する転移酵素（EC 2）である。

配列類似性。ECカテゴリーは配列の類似性を反映しない。例えば、全く同じ反応を触媒する同じEC番号の2つのリガーゼが、全く異なる配列を持つことがある。酵素はその機能とは無関係に、他のタンパク質と同様に、配列の類似性によって数多くのファミリーに分類されている。これらのファミリーは、Pfamのような何十種類ものタンパク質やタンパク質ファミリーのデータベースに記録されている。

Non-homologous isofunctional enzymes. Unrelated enzymes that have the same enzymatic activity have been called non-homologous isofunctional enzymes. Horizontal gene transfer may spread these genes to unrelated species, especially bacteria where they can replace endogenous genes of the same function, leading to hon-homologous gene displacement.

Structure

Enzyme activity initially increases with temperature (Q10 coefficient) until the enzyme's structure unfolds (denaturation), leading to an optimal rate of reaction at an intermediate temperature.

See also: Protein structure

Enzymes are generally globular proteins, acting alone or in larger complexes. The sequence of the amino acids specifies the structure which in turn determines the catalytic activity of the enzyme. Although structure determines function, a novel enzymatic activity cannot yet be predicted from structure alone. Enzyme structures unfold (denature) when heated or exposed to chemical denaturants and this disruption to the structure typically causes a loss of activity. Enzyme denaturation is normally linked to temperatures above a species' normal level; as a result, enzymes from bacteria living in volcanic environments such as hot springs are prized by industrial users for their ability to function at high temperatures, allowing enzyme-catalysed reactions to be operated at a very high rate.

Enzymes are usually much larger than their substrates. Sizes range from just 62 amino acid residues, for the monomer of 4-oxalocrotonate tautomerase, to over 2,500 residues in the animal fatty acid synthase. Only a small portion of their structure (around 2–4 amino acids) is directly involved in catalysis: the catalytic site. This catalytic site is located next to one or more binding sites where residues orient the substrates. The catalytic site and binding site together compose the enzyme's active site. The remaining majority of the enzyme structure serves to maintain the precise orientation and dynamics of the active site.

In some enzymes, no amino acids are directly involved in catalysis; instead, the enzyme contains sites to bind and orient catalytic cofactors. Enzyme structures may also contain allosteric sites where the binding of a small molecule causes a conformational change that increases or decreases activity.

A small number of RNA-based biological catalysts called ribozymes exist, which again can act alone or in complex with proteins. The most common of these is the ribosome which is a complex of protein and catalytic RNA components.

Mechanism

Organisation of enzyme structure and lysozyme example. Binding sites in blue, catalytic site in red and peptidoglycan substrate in black. (PDB: 9LYZ​)

Substrate binding

Enzymes must bind their substrates before they can catalyse any chemical reaction. Enzymes are usually very specific as to what substrates they bind and then the chemical reaction catalysed. Specificity is achieved by binding pockets with complementary shape, charge and hydrophilic/hydrophobic characteristics to the substrates. Enzymes can therefore distinguish between very similar substrate molecules to be chemoselective, regioselective and stereospecific.

Some of the enzymes showing the highest specificity and accuracy are involved in the copying and expression of the genome. Some of these enzymes have "proof-reading" mechanisms. Here, an enzyme such as DNA polymerase catalyzes a reaction in a first step and then checks that the product is correct in a second step. This two-step process results in average error rates of less than 1 error in 100 million reactions in high-fidelity mammalian polymerases. aminoacyl tRNA synthetases and ribosomes.

Conversely, some enzymes display enzyme promiscuity, having broad specificity and acting on a range of different physiologically relevant substrates. Many enzymes possess small side activities which arose fortuitously (i.e. neutrally), which may be the starting point for the evolutionary selection of a new function.

Enzyme changes shape by induced fit upon substrate binding to form enzyme-substrate complex. Hexokinase has a large induced fit motion that closes over the substrates adenosine triphosphate and xylose. Binding sites in blue, substrates in black and Mg²⁺ cofactor in yellow. (PDB: 2E2N​, 2E2Q​)

"Lock and key" model

To explain the observed specificity of enzymes, in 1894 Emil Fischer proposed that both the enzyme and the substrate possess specific complementary geometric shapes that fit exactly into one another. This is often referred to as "the lock and key" model. This early model explains enzyme specificity, but fails to explain the stabilization of the transition state that enzymes achieve.

Induced fit model

In 1958, Daniel Koshland suggested a modification to the lock and key model: since enzymes are rather flexible structures, the active site is continuously reshaped by interactions with the substrate as the substrate interacts with the enzyme. As a result, the substrate does not simply bind to a rigid active site; the amino acid side-chains that make up the active site are molded into the precise positions that enable the enzyme to perform its catalytic function. In some cases, such as glycosidases, the substrate molecule also changes shape slightly as it enters the active site. The active site continues to change until the substrate is completely bound, at which point the final shape and charge distribution is determined. Induced fit may enhance the fidelity of molecular recognition in the presence of competition and noise via the conformational proofreading mechanism.

Catalysis

See also: Enzyme catalysis and Transition state theory

Enzymes can accelerate reactions in several ways, all of which lower the activation energy (ΔG^‡, Gibbs free energy)

1. By stabilizing the transition state:
   • Creating an environment with a charge distribution complementary to that of the transition state to lower its energy
2. By providing an alternative reaction pathway:
   • Temporarily reacting with the substrate, forming a covalent intermediate to provide a lower energy transition state
3. By destabilising the substrate ground state:
   • Distorting bound substrate(s) into their transition state form to reduce the energy required to reach the transition state
   • By orienting the substrates into a productive arrangement to reduce the reaction entropy change (the contribution of this mechanism to catalysis is relatively small)

Enzymes may use several of these mechanisms simultaneously. For example, proteases such as trypsin perform covalent catalysis using a catalytic triad, stabilise charge build-up on the transition states using an oxyanion hole, complete hydrolysis using an oriented water substrate.

Dynamics

See also: Protein dynamics

Enzymes are not rigid, static structures; instead they have complex internal dynamic motions – that is, movements of parts of the enzyme's structure such as individual amino acid residues, groups of residues forming a protein loop or unit of secondary structure, or even an entire protein domain. These motions give rise to a conformational ensemble of slightly different structures that interconvert with one another at equilibrium. Different states within this ensemble may be associated with different aspects of an enzyme's function. For example, different conformations of the enzyme dihydrofolate reductase are associated with the substrate binding, catalysis, cofactor release, and product release steps of the catalytic cycle, consistent with catalytic resonance theory.

Substrate presentation

Substrate presentation is a process where the enzyme is sequestered away from its substrate. Enzymes can be sequestered to the plasma membrane away from a substrate in the nucleus or cytosol. Or within the membrane, an enzyme can be sequestered into lipid rafts away from its substrate in the disordered region. When the enzyme is released it mixes with its substrate. Alternatively, the enzyme can be sequestered near its substrate to activate the enzyme. For example, the enzyme can be soluble and upon activation bind to a lipid in the plasma membrane and then act upon molecules in the plasma membrane.

Allosteric modulation

Main article: Allosteric regulation

Allosteric sites are pockets on the enzyme, distinct from the active site, that bind to molecules in the cellular environment. These molecules then cause a change in the conformation or dynamics of the enzyme that is transduced to the active site and thus affects the reaction rate of the enzyme. In this way, allosteric interactions can either inhibit or activate enzymes. Allosteric interactions with metabolites upstream or downstream in an enzyme's metabolic pathway cause feedback regulation, altering the activity of the enzyme according to the flux through the rest of the pathway.

Cofactors

Chemical structure for thiamine pyrophosphate and protein structure of transketolase. Thiamine pyrophosphate cofactor in yellow and xylulose 5-phosphate substrate in black. (PDB: 4KXV​)

Main article: Cofactor (biochemistry)

Some enzymes do not need additional components to show full activity. Others require non-protein molecules called cofactors to be bound for activity. Cofactors can be either inorganic (e.g., metal ions and iron–sulfur clusters) or organic compounds (e.g., flavin and heme). These cofactors serve many purposes; for instance, metal ions can help in stabilizing nucleophilic species within the active site. Organic cofactors can be either coenzymes, which are released from the enzyme's active site during the reaction, or prosthetic groups, which are tightly bound to an enzyme. Organic prosthetic groups can be covalently bound (e.g., biotin in enzymes such as pyruvate carboxylase).

An example of an enzyme that contains a cofactor is carbonic anhydrase, which uses a zinc cofactor bound as part of its active site. These tightly bound ions or molecules are usually found in the active site and are involved in catalysis. For example, flavin and heme cofactors are often involved in redox reactions.

Enzymes that require a cofactor but do not have one bound are called apoenzymes or apoproteins. An enzyme together with the cofactor(s) required for activity is called a holoenzyme (or haloenzyme). The term holoenzyme can also be applied to enzymes that contain multiple protein subunits, such as the DNA polymerases; here the holoenzyme is the complete complex containing all the subunits needed for activity.

Coenzymes

Coenzymes are small organic molecules that can be loosely or tightly bound to an enzyme. Coenzymes transport chemical groups from one enzyme to another. Examples include NADH, NADPH and adenosine triphosphate (ATP). Some coenzymes, such as flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), thiamine pyrophosphate (TPP), and tetrahydrofolate (THF), are derived from vitamins. These coenzymes cannot be synthesized by the body de novo and closely related compounds (vitamins) must be acquired from the diet. The chemical groups carried include:

• the hydride ion (H⁻), carried by NAD or NADP⁺
• the phosphate group, carried by adenosine triphosphate
• the acetyl group, carried by coenzyme A
• formyl, methenyl or methyl groups, carried by folic acid and
• the methyl group, carried by S-adenosylmethionine

Since coenzymes are chemically changed as a consequence of enzyme action, it is useful to consider coenzymes to be a special class of substrates, or second substrates, which are common to many different enzymes. For example, about 1000 enzymes are known to use the coenzyme NADH.

Coenzymes are usually continuously regenerated and their concentrations maintained at a steady level inside the cell. For example, NADPH is regenerated through the pentose phosphate pathway and S-adenosylmethionine by methionine adenosyltransferase. This continuous regeneration means that small amounts of coenzymes can be used very intensively. For example, the human body turns over its own weight in ATP each day.

Thermodynamics

The energies of the stages of a chemical reaction. Uncatalysed (dashed line), substrates need a lot of activation energy to reach a transition state, which then decays into lower-energy products. When enzyme catalysed (solid line), the enzyme binds the substrates (ES), then stabilizes the transition state (ES^‡) to reduce the activation energy required to produce products (EP) which are finally released.

Main articles: Activation energy, Thermodynamic equilibrium, and Chemical equilibrium

As with all catalysts, enzymes do not alter the position of the chemical equilibrium of the reaction. In the presence of an enzyme, the reaction runs in the same direction as it would without the enzyme, just more quickly. For example, carbonic anhydrase catalyzes its reaction in either direction depending on the concentration of its reactants:

$\mathrm{C}\mathrm{O}{\phantom{A}}_2+\mathrm{H}{\phantom{A}}_2\mathrm{O}\stackrel{\text{Carbonic anhydrase}}{\to }\mathrm{H}{\phantom{A}}_2\mathrm{C}\mathrm{O}{\phantom{A}}_3$ (in tissues; high CO₂ concentration)

 

 

 

 

(1)

$\mathrm{C}\mathrm{O}{\phantom{A}}_2+\mathrm{H}{\phantom{A}}_2\mathrm{O}\stackrel{\text{Carbonic anhydrase}}{\leftarrow }\mathrm{H}{\phantom{A}}_2\mathrm{C}\mathrm{O}{\phantom{A}}_3$ (in lungs; low CO₂ concentration)

 

 

 

 

(2)

The rate of a reaction is dependent on the activation energy needed to form the transition state which then decays into products. Enzymes increase reaction rates by lowering the energy of the transition state. First, binding forms a low energy enzyme-substrate complex (ES). Second, the enzyme stabilises the transition state such that it requires less energy to achieve compared to the uncatalyzed reaction (ES^‡). Finally the enzyme-product complex (EP) dissociates to release the products.

Enzymes can couple two or more reactions, so that a thermodynamically favorable reaction can be used to "drive" a thermodynamically unfavourable one so that the combined energy of the products is lower than the substrates. For example, the hydrolysis of ATP is often used to drive other chemical reactions.

Kinetics

A chemical reaction mechanism with or without enzyme catalysis. The enzyme (E) binds substrate (S) to produce product (P).

Saturation curve for an enzyme reaction showing the relation between the substrate concentration and reaction rate.

Main article: Enzyme kinetics

Enzyme kinetics is the investigation of how enzymes bind substrates and turn them into products. The rate data used in kinetic analyses are commonly obtained from enzyme assays. In 1913 Leonor Michaelis and Maud Leonora Menten proposed a quantitative theory of enzyme kinetics, which is referred to as Michaelis–Menten kinetics. The major contribution of Michaelis and Menten was to think of enzyme reactions in two stages. In the first, the substrate binds reversibly to the enzyme, forming the enzyme-substrate complex. This is sometimes called the Michaelis–Menten complex in their honor. The enzyme then catalyzes the chemical step in the reaction and releases the product. This work was further developed by G. E. Briggs and J. B. S. Haldane, who derived kinetic equations that are still widely used today.

Enzyme rates depend on solution conditions and substrate concentration. To find the maximum speed of an enzymatic reaction, the substrate concentration is increased until a constant rate of product formation is seen. This is shown in the saturation curve on the right. Saturation happens because, as substrate concentration increases, more and more of the free enzyme is converted into the substrate-bound ES complex. At the maximum reaction rate (V_max) of the enzyme, all the enzyme active sites are bound to substrate, and the amount of ES complex is the same as the total amount of enzyme.

V_max is only one of several important kinetic parameters. The amount of substrate needed to achieve a given rate of reaction is also important. This is given by the Michaelis–Menten constant (K_m), which is the substrate concentration required for an enzyme to reach one-half its maximum reaction rate; generally, each enzyme has a characteristic K_M for a given substrate. Another useful constant is k_cat, also called the turnover number, which is the number of substrate molecules handled by one active site per second.

The efficiency of an enzyme can be expressed in terms of k_cat/K_m. This is also called the specificity constant and incorporates the rate constants for all steps in the reaction up to and including the first irreversible step. Because the specificity constant reflects both affinity and catalytic ability, it is useful for comparing different enzymes against each other, or the same enzyme with different substrates. The theoretical maximum for the specificity constant is called the diffusion limit and is about 10⁸ to 10⁹ (M⁻¹ s⁻¹). At this point every collision of the enzyme with its substrate will result in catalysis, and the rate of product formation is not limited by the reaction rate but by the diffusion rate. Enzymes with this property are called catalytically perfect or kinetically perfect. Example of such enzymes are triose-phosphate isomerase, carbonic anhydrase, acetylcholinesterase, catalase, fumarase, β-lactamase, and superoxide dismutase. The turnover of such enzymes can reach several million reactions per second. But most enzymes are far from perfect: the average values of $k_{\mathrm{c}\mathrm{a}\mathrm{t}}/K_{\mathrm{m}}$ and $k_{\mathrm{c}\mathrm{a}\mathrm{t}}$ are about $10^5{\mathrm{s}}^{-1}{\mathrm{M}}^{-1}$ and $10{\mathrm{s}}^{-1}$, respectively.

Michaelis–Menten kinetics relies on the law of mass action, which is derived from the assumptions of free diffusion and thermodynamically driven random collision. Many biochemical or cellular processes deviate significantly from these conditions, because of macromolecular crowding and constrained molecular movement. More recent, complex extensions of the model attempt to correct for these effects.

Inhibition

The coenzyme folic acid (left) and the anti-cancer drug methotrexate (right) are very similar in structure (differences show in green). As a result, methotrexate is a competitive inhibitor of many enzymes that use folates.

Main article: Enzyme inhibitor

Enzyme reaction rates can be decreased by various types of enzyme inhibitors.

Types of inhibition

Competitive

A competitive inhibitor and substrate cannot bind to the enzyme at the same time. Often competitive inhibitors strongly resemble the real substrate of the enzyme. For example, the drug methotrexate is a competitive inhibitor of the enzyme dihydrofolate reductase, which catalyzes the reduction of dihydrofolate to tetrahydrofolate. The similarity between the structures of dihydrofolate and this drug are shown in the accompanying figure. This type of inhibition can be overcome with high substrate concentration. In some cases, the inhibitor can bind to a site other than the binding-site of the usual substrate and exert an allosteric effect to change the shape of the usual binding-site.

Non-competitive

A non-competitive inhibitor binds to a site other than where the substrate binds. The substrate still binds with its usual affinity and hence K_m remains the same. However the inhibitor reduces the catalytic efficiency of the enzyme so that V_max is reduced. In contrast to competitive inhibition, non-competitive inhibition cannot be overcome with high substrate concentration.

Uncompetitive

An uncompetitive inhibitor cannot bind to the free enzyme, only to the enzyme-substrate complex; hence, these types of inhibitors are most effective at high substrate concentration. In the presence of the inhibitor, the enzyme-substrate complex is inactive. This type of inhibition is rare.

Mixed

A mixed inhibitor binds to an allosteric site and the binding of the substrate and the inhibitor affect each other. The enzyme's function is reduced but not eliminated when bound to the inhibitor. This type of inhibitor does not follow the Michaelis–Menten equation.

Irreversible

An irreversible inhibitor permanently inactivates the enzyme, usually by forming a covalent bond to the protein. Penicillin and aspirin are common drugs that act in this manner.

Functions of inhibitors

In many organisms, inhibitors may act as part of a feedback mechanism. If an enzyme produces too much of one substance in the organism, that substance may act as an inhibitor for the enzyme at the beginning of the pathway that produces it, causing production of the substance to slow down or stop when there is sufficient amount. This is a form of negative feedback. Major metabolic pathways such as the citric acid cycle make use of this mechanism.

Since inhibitors modulate the function of enzymes they are often used as drugs. Many such drugs are reversible competitive inhibitors that resemble the enzyme's native substrate, similar to methotrexate above; other well-known examples include statins used to treat high cholesterol, and protease inhibitors used to treat retroviral infections such as HIV. A common example of an irreversible inhibitor that is used as a drug is aspirin, which inhibits the COX-1 and COX-2 enzymes that produce the inflammation messenger prostaglandin. Other enzyme inhibitors are poisons. For example, the poison cyanide is an irreversible enzyme inhibitor that combines with the copper and iron in the active site of the enzyme cytochrome c oxidase and blocks cellular respiration.

Factors affecting enzyme activity

As enzymes are made up of proteins, their actions are sensitive to change in many physio chemical factors such as pH, temperature, substrate concentration, etc.

The following table shows pH optima for various enzymes.

Enzyme	Optimum pH	pH description
Pepsin	1.5–1.6	Highly acidic
Invertase	4.5	Acidic
Lipase (stomach)	4.0–5.0	Acidic
Lipase (castor oil)	4.7	Acidic
Lipase (pancreas)	8.0	Alkaline
Amylase (malt)	4.6–5.2	Acidic
Amylase (pancreas)	6.7–7.0	Acidic-neutral
Cellobiase	5.0	Acidic
Maltase	6.1–6.8	Acidic
Sucrase	6.2	Acidic
Catalase	7.0	Neutral
Urease	7.0	Neutral
Cholinesterase	7.0	Neutral
Ribonuclease	7.0–7.5	Neutral
Fumarase	7.8	Alkaline
Trypsin	7.8–8.7	Alkaline
Adenosine triphosphate	9.0	Alkaline
Arginase	10.0	Highly alkaline

Biological function

Enzymes serve a wide variety of functions inside living organisms. They are indispensable for signal transduction and cell regulation, often via kinases and phosphatases. They also generate movement, with myosin hydrolyzing adenosine triphosphate (ATP) to generate muscle contraction, and also transport cargo around the cell as part of the cytoskeleton. Other ATPases in the cell membrane are ion pumps involved in active transport. Enzymes are also involved in more exotic functions, such as luciferase generating light in fireflies. Viruses can also contain enzymes for infecting cells, such as the HIV integrase and reverse transcriptase, or for viral release from cells, like the influenza virus neuraminidase.

An important function of enzymes is in the digestive systems of animals. Enzymes such as amylases and proteases break down large molecules (starch or proteins, respectively) into smaller ones, so they can be absorbed by the intestines. Starch molecules, for example, are too large to be absorbed from the intestine, but enzymes hydrolyze the starch chains into smaller molecules such as maltose and eventually glucose, which can then be absorbed. Different enzymes digest different food substances. In ruminants, which have herbivorous diets, microorganisms in the gut produce another enzyme, cellulase, to break down the cellulose cell walls of plant fiber.

Metabolism

The metabolic pathway of glycolysis releases energy by converting glucose to pyruvate via a series of intermediate metabolites. Each chemical modification (red box) is performed by a different enzyme.

Several enzymes can work together in a specific order, creating metabolic pathways. In a metabolic pathway, one enzyme takes the product of another enzyme as a substrate. After the catalytic reaction, the product is then passed on to another enzyme. Sometimes more than one enzyme can catalyze the same reaction in parallel; this can allow more complex regulation: with, for example, a low constant activity provided by one enzyme but an inducible high activity from a second enzyme.

Enzymes determine what steps occur in these pathways. Without enzymes, metabolism would neither progress through the same steps and could not be regulated to serve the needs of the cell. Most central metabolic pathways are regulated at a few key steps, typically through enzymes whose activity involves the hydrolysis of ATP. Because this reaction releases so much energy, other reactions that are thermodynamically unfavorable can be coupled to ATP hydrolysis, driving the overall series of linked metabolic reactions.

Control of activity

There are five main ways that enzyme activity is controlled in the cell.

Regulation

Enzymes can be either activated or inhibited by other molecules. For example, the end product(s) of a metabolic pathway are often inhibitors for one of the first enzymes of the pathway (usually the first irreversible step, called committed step), thus regulating the amount of end product made by the pathways. Such a regulatory mechanism is called a negative feedback mechanism, because the amount of the end product produced is regulated by its own concentration. Negative feedback mechanism can effectively adjust the rate of synthesis of intermediate metabolites according to the demands of the cells. This helps with effective allocations of materials and energy economy, and it prevents the excess manufacture of end products. Like other homeostatic devices, the control of enzymatic action helps to maintain a stable internal environment in living organisms.

Post-translational modification

Examples of post-translational modification include phosphorylation, myristoylation and glycosylation. For example, in the response to insulin, the phosphorylation of multiple enzymes, including glycogen synthase, helps control the synthesis or degradation of glycogen and allows the cell to respond to changes in blood sugar. Another example of post-translational modification is the cleavage of the polypeptide chain. Chymotrypsin, a digestive protease, is produced in inactive form as chymotrypsinogen in the pancreas and transported in this form to the stomach where it is activated. This stops the enzyme from digesting the pancreas or other tissues before it enters the gut. This type of inactive precursor to an enzyme is known as a zymogen or proenzyme.

Quantity

Enzyme production (transcription and translation of enzyme genes) can be enhanced or diminished by a cell in response to changes in the cell's environment. This form of gene regulation is called enzyme induction. For example, bacteria may become resistant to antibiotics such as penicillin because enzymes called beta-lactamases are induced that hydrolyse the crucial beta-lactam ring within the penicillin molecule. Another example comes from enzymes in the liver called cytochrome P450 oxidases, which are important in drug metabolism. Induction or inhibition of these enzymes can cause drug interactions. Enzyme levels can also be regulated by changing the rate of enzyme degradation. The opposite of enzyme induction is enzyme repression.

Subcellular distribution

Enzymes can be compartmentalized, with different metabolic pathways occurring in different cellular compartments. For example, fatty acids are synthesized by one set of enzymes in the cytosol, endoplasmic reticulum and Golgi and used by a different set of enzymes as a source of energy in the mitochondrion, through β-oxidation. In addition, trafficking of the enzyme to different compartments may change the degree of protonation (e.g., the neutral cytoplasm and the acidic lysosome) or oxidative state (e.g., oxidizing periplasm or reducing cytoplasm) which in turn affects enzyme activity. In contrast to partitioning into membrane bound organelles, enzyme subcellular localisation may also be altered through polymerisation of enzymes into macromolecular cytoplasmic filaments.

Organ specialization

In multicellular eukaryotes, cells in different organs and tissues have different patterns of gene expression and therefore have different sets of enzymes (known as isozymes) available for metabolic reactions. This provides a mechanism for regulating the overall metabolism of the organism. For example, hexokinase, the first enzyme in the glycolysis pathway, has a specialized form called glucokinase expressed in the liver and pancreas that has a lower affinity for glucose yet is more sensitive to glucose concentration. This enzyme is involved in sensing blood sugar and regulating insulin production.

Involvement in disease

In phenylalanine hydroxylase over 300 different mutations throughout the structure cause phenylketonuria. Phenylalanine substrate and tetrahydrobiopterin coenzyme in black, and Fe²⁺ cofactor in yellow. (PDB: 1KW0​)

Hereditary defects in enzymes are generally inherited in an autosomal fashion because there are more non-X chromosomes than X-chromosomes, and a recessive fashion because the enzymes from the unaffected genes are generally sufficient to prevent symptoms in carriers.

See also: Genetic disorder

Since the tight control of enzyme activity is essential for homeostasis, any malfunction (mutation, overproduction, underproduction or deletion) of a single critical enzyme can lead to a genetic disease. The malfunction of just one type of enzyme out of the thousands of types present in the human body can be fatal. An example of a fatal genetic disease due to enzyme insufficiency is Tay–Sachs disease, in which patients lack the enzyme hexosaminidase.

One example of enzyme deficiency is the most common type of phenylketonuria. Many different single amino acid mutations in the enzyme phenylalanine hydroxylase, which catalyzes the first step in the degradation of phenylalanine, result in build-up of phenylalanine and related products. Some mutations are in the active site, directly disrupting binding and catalysis, but many are far from the active site and reduce activity by destabilising the protein structure, or affecting correct oligomerisation. This can lead to intellectual disability if the disease is untreated. Oral administration of enzymes can be used to treat some functional enzyme deficiencies, such as pancreatic insufficiency and lactose intolerance.

Another way enzyme malfunctions can cause disease comes from germline mutations in genes coding for DNA repair enzymes. Defects in these enzymes cause cancer because cells are less able to repair mutations in their genomes. This causes a slow accumulation of mutations and results in the development of cancers. An example of such a hereditary cancer syndrome is xeroderma pigmentosum, which causes the development of skin cancers in response to even minimal exposure to ultraviolet light.

Evolution

Similar to any other protein, enzymes change over time through mutations and sequence divergence. Given their central role in metabolism, enzyme evolution plays a critical role in adaptation. A key question is therefore whether and how enzymes can change their enzymatic activities alongside. It is generally accepted that many new enzyme activities have evolved through gene duplication and mutation of the duplicate copies although evolution can also happen without duplication. One example of an enzyme that has changed its activity is the ancestor of methionyl aminopeptidase (MAP) and creatine amidinohydrolase (creatinase) which are clearly homologous but catalyze very different reactions (MAP removes the amino-terminal methionine in new proteins while creatinase hydrolyses creatine to sarcosine and urea). In addition, MAP is metal-ion dependent while creatinase is not, hence this property was also lost over time. Small changes of enzymatic activity are extremely common among enzymes. In particular, substrate binding specificity (see above) can easily and quickly change with single amino acid changes in their substrate binding pockets. This is frequently seen in the main enzyme classes such as kinases.

Artificial (in vitro) evolution is now commonly used to modify enzyme activity or specificity for industrial applications (see below).

Industrial applications

Main article: Industrial enzymes

Enzymes are used in the chemical industry and other industrial applications when extremely specific catalysts are required. Enzymes in general are limited in the number of reactions they have evolved to catalyze and also by their lack of stability in organic solvents and at high temperatures. As a consequence, protein engineering is an active area of research and involves attempts to create new enzymes with novel properties, either through rational design or in vitro evolution. These efforts have begun to be successful, and a few enzymes have now been designed "from scratch" to catalyze reactions that do not occur in nature.

Application	Enzymes used	Uses
Biofuel industry	Cellulases	Break down cellulose into sugars that can be fermented to produce cellulosic ethanol.
	Ligninases	Pretreatment of biomass for biofuel production.
Biological detergent	Proteases, amylases, lipases	Remove protein, starch, and fat or oil stains from laundry and dishware.
	Mannanases	Remove food stains from the common food additive guar gum.
Brewing industry	Amylase, glucanases, proteases	Split polysaccharides and proteins in the malt.
	Betaglucanases	Improve the wort and beer filtration characteristics.
	Amyloglucosidase and pullulanases	Make low-calorie beer and adjust fermentability.
	Acetolactate decarboxylase (ALDC)	Increase fermentation efficiency by reducing diacetyl formation.
Culinary uses	Papain	Tenderize meat for cooking.
Dairy industry	Rennin	Hydrolyze protein in the manufacture of cheese.
	Lipases	Produce Camembert cheese and blue cheeses such as Roquefort.
Food processing	Amylases	Produce sugars from starch, such as in making high-fructose corn syrup.
	Proteases	Lower the protein level of flour, as in biscuit-making.
	Trypsin	Manufacture hypoallergenic baby foods.
	Cellulases, pectinases	Clarify fruit juices.
Molecular biology	Nucleases, DNA ligase and polymerases	Use restriction digestion and the polymerase chain reaction to create recombinant DNA.
Paper industry	Xylanases, hemicellulases and lignin peroxidases	Remove lignin from kraft pulp.
Personal care	Proteases	Remove proteins on contact lenses to prevent infections.
Starch industry	Amylases	Convert starch into glucose and various syrups.

See also

• Industrial enzymes
• List of enzymes
• Molecular machine

Enzyme databases

• BRENDA
• ExPASy
• IntEnz
• KEGG
• MetaCyc

Further reading

General

• Berg JM, Tymoczko JL, Stryer L (2002). Biochemistry (5th ed.). New York, NY: W. H. Freeman. ISBN 0-7167-3051-0., A biochemistry textbook available free online through NCBI Bookshelf.

Etymology and history

• Cornish-Bowden A, ed. (1997). New Beer in an Old Bottle: Eduard Buchner and the Growth of Biochemical Knowledge. Universitat de València. ISBN 84-370-3328-4. Archived from the original on 13 December 2010. Retrieved 27 June 2006., A history of early enzymology.

Enzyme structure and mechanism

• Suzuki H (2015). How Enzymes Work: From Structure to Function. Boca Raton, FL: CRC Press. ISBN 978-981-4463-92-8.

Kinetics and inhibition

• Cornish-Bowden A (2012). Fundamentals of Enzyme Kinetics (4th ed.). Weinheim: Wiley-VCH. ISBN 978-3527330744.

External links

• Media related to Enzymes at Wikimedia Commons