Chapter 3. Enzyme In Book: Chapter 5 Properties of Enzymes Chapter 6 Mechanisms of Enzymes

Lecture 1 Introduction Lecture 2 Kinetics of enzymatic reactions Lecture 3 Mechanism of enzymes – catalyzed reaction Lecture 4 Regulation of enzyme activities

Lecture 1. Introduction

In 1850s, Louis Pasteur concluded that fermentation of sugar into alcohol by yeast is catalyzed by “ferments” . 【发酵】【酒精】【酵母】【发酵酶】Louis Pasteur :Louis Pasteur was a French chemist best known for his remarkable breakthroughs in microbiology. His experiments countered the common view of spontaneous generation and confirmed the germ theory of disease, and he created the first vaccine for rabies. Pasteur is best known to the general public for describing how to stop milk and wine from going sour: This process came to be called pasteurization. (1822～1895)

Frederick W. Kühne called these molecules enzymes. Then in 1897, Eduard Buchner discovered that yeast extracts could ferment sugar to alcohol, proving that the presence of living yeast cells was not needed for fermentation. Frederick W. Kühne called these molecules enzymes. 【发酵】【酒精】【酵母】【发酵酶】

General properties of enzymes Enzymes are extraordinarily efficient, selective, biological catalysts. The primary role of enzymes is to enhance the rates of these reactions by lowering the amount of energy needed for reactions to occur.

Figure 6.1 Energy diagram for a single-step reaction. transition Figure 6.1 Energy diagram for a single-step reaction.

General properties of enzymes cont’ Enzymes are unchanged in the overall process; It recycles to participate in multiple reactions. E+S→ES→E+P Enzymes are highly specific for reactants or substrates. - Acts on a single substrates or group of related substrates - Many exhibit stereospecificity

Stereospecificity in substrate binding.

General properties of enzymes cont’ Almost all enzymes are proteins.(exception: ribozymes) Higher reaction rates. 103 to 1020 over uncatalyzed rates. Milder reaction conditions. Capacity for regulation.

Catalysis and chemical nature of enzymes Enzyme —— any naturally occurring or synthetic macromolecular substance composed wholly or largely of protein, that catalysts, more or less specifically, one or more (bio)chemical reactions at relatively low temperatures. 酶是活细胞产生的一类具有催化功能的生物分子，又称为生物催化剂（Biocatalysts）。

Catalytic Power Enzymes can accelerate reactions as much as 1016 over uncatalyzed rates!

Specificity: ES Complex Active site Hydrophobic H-bonds Ionic Interactoins

Conjugated enzyme & Cofactor Inactive Simple enzyme Enzyme Apoenzyme Metal ion Conjugated enzyme Cofactor Small organic molecules …… Holoenzyme Active 【单纯酶】【结合酶】【脱辅酶】【辅因子】有机小分子【全酶】 【金属离子】

（细胞色素氧化酶）（丙酮酸激酶）

Monomeric enzyme, oligomeric enzyme & multienzyme complex —— Any multi-enzyme in which catalytic domains occur on more than one type of polypeptide chain. 单体酶（只由1条肽链构成。为数不多，全部为水解酶。 寡聚酶（由2个以上的亚基组成，亚基间非共价结合，解离则失活。 （多酶络合物）由2种以上功能相关的酶嵌合而成的酶分子，分子量大，有利于系列化学反应的连续进行，提高催化效率。

Pyruvate dehydrogenase system 【丙酮酸脱氢酶系】硫辛酸乙酰转移酶二氢硫辛酸脱氢酶丙酮酸脱羧酶

Active site and essential group Active center The general region of an enzyme molecule containing the catalytic residues identified with the binding and reaction of substrate (s). Binding site Catalytic site （活性中心）指酶分子中直接和底物结合，并催化底物产生反应的部位。 包括结合部位和催化部位

Which amino acids can be in active center? Ser、Thr、His、Cys、Asp、Glu、Lys

Active center groups Ser-OH Cys-SH His-imidazolyl 【咪唑基】

Tyr- phenolic hydroxyl group Asp-COOH Glu-COOH Lys-NH2 Tyr- phenolic hydroxyl group 【酚羟基】

【胰凝乳蛋白酶】

The activation of zymogen Zymogen (or proenzyme） —— The inactive precursor of an enzyme, often convertible to the enzyme by partial proteolysis. (酶原) 【蛋白酶解】在细胞内合成及分泌的没有活性的酶的前体。

Zymogen activation —— Zymogen only acquires full activity upon specific proteolytic cleavage of one or several of their peptide bonds. （酶原的激活） 酶原分子内局部肽键断裂，分子构象发生一定程度改变，从而形成或暴露酶的活性中心的过程。 酶原激活存在级联反应（cascade reaction）。

Isoenzyme Isoenzyme or isozyme —— Any one of the multiple forms of an enzyme arising from a genetically determined difference in primary structure. Lactate dehydrogenase, LDH （同工酶）指能催化相同的化学反应，但分子结构、组成却有所不同的一组酶。 一般为寡聚蛋白。 举例： 乳酸脱氢酶（

Lactate Dehydrogenase (LDH) It is formed by the association of four peptide chains of two different kinds of monomers: M and H The variants seen in humans are:  LDH1: M M M M  LDH2: M M M H LDH3: M M H H  LDH4: M H H H   LDH5: H H H H

同工酶是指催化相同的化学反应，而酶蛋白的分子结构、理化性质以及免疫学性质不同的一组酶。他们是由不同基因或等位基因编码的多肽链，或由同一基因转录生成的不同mRNA翻译的不同多肽链组成的蛋白质。同工酶存在与同一种属或同一个体的不同组织或同一细胞的不同亚细胞结构中，它在代谢调节上起着重要的作用。研究最的同工酶是乳酸脱氢酶(LDH)。 　　乳酸脱氢酶同工酶(LDHs)为四聚体，在体内共有五种分子形式，即LDH1(H4)，LDH2(H3M)，LDH3(H2M2)，LDH4(HM3)和LDH5(M4)。 　　心肌中以LDH1含量最多，LDH1对乳酸的亲和力较高，因此它的主要作用是催化乳酸转变为丙酮酸再进一步氧化分解，以供应心肌的能量。在骨骼肌中含量最多的是LDH5，LDH5对丙酮酸的亲和力较高，因此它的主要作用是催化丙酮酸转变为乳酸，以促进糖酵解的进行。LDH1: M M M M (abundant in heart, brain erythrocytes; around 33% of serum LDH)   LDH2: M M M H (abundant in heart, brain erythrocytes; around 45% of serum LDH) LDH3: M M H H (abundant in brain, kidneys, lung; around 18 % of serum LDH) LDH4: M H H H ((abundant in liver, skeletal muscle, kidney; around 3% of serum LDH) LDH5: H H H H ((abundant in liver, skeletal muscle, ileum; around 1 % of serum LDH)

Mechanisms of specificity lock and key theory induced-fit theory （锁钥学说）：认为酶分子的天然构象是刚性的结构，酶的活性中心具有特定的形状，与底物的结合如同一把钥匙对一把锁一样互补。 （诱导契合学说）：认为酶活性中心与底物结构并非完全互补，但具有一定的柔性，由于底物的诱导才形成了互补形状，从而有利于底物的结合。

a. Lock-and-key theory Both E and S are rigid and fixed, so they must be complementary to each other perfectly in order to have a right match. （锁钥学说）

b. induced-fit hypothesis The binding induces conformational changes of both E and S, forcing them to get a perfect match. ( “诱导契合”假说) 【构象】

Enzyme Classification Adding the suffix–ase to the substrate end -Urea: Urease Descriptive term for the reactions they catalyze -Alcohol dehydrogenase: catalyzes the removal of hydrogen from alcohols Historic names: Trypsin

Classes of enzymes EC number introduced by a committee of the International Union of Biochemistry and Molecular Biology (IUBMB)

The Six Classes of Enzymes Classification Type of Reaction Catalyzed 1. Oxidoreductases Oxidation–reduction reactions 2. Transferases Transfer of functional groups 3. Hydrolases Hydrolysis reactions 4. Lyases Group elimination to form double bonds 5. Isomerases Isomerization 6. Ligases Bond formation coupled with ATP hydrolysis

Oxidoreductases Dehydrogenase, Oxidase, Peroxidases, Reductases, Monooxygenases, Dioxygenases 【氧化还原酶类】 The oxidoreductases (class 1) catalyze the transfer of reducing equivalents(Hydrogen and electrons)from one redox system to another.

Transferases C1-Trasferases, Glycosyltransferases, Aminotransferases, Phosphotransferases 【转移酶类】催化基团转移反应。 如谷丙转氨酶催化的氨基转移反应。 The transferases (class 2) catalyze the transfer of other groups from one molecule to another. Oxidoreductases and transferases generally require coenzymes

Hydrolases Esterases, Glycosidases, Peptidases, Amidases 【水解酶类】催化底物的加水分解反应。 包括淀粉酶、蛋白酶、核酸酶及脂酶等。The hydrolases (class 3) hydrolases cause cleavage of bond using water

Lyases C-C-Lyases, C-O-Lyases, C-N-Lyases, C-S-Lyases 【裂合（裂解）酶类】催化从底物分子中移去基团或原子而形成双键的反应及其逆反应。 包括醛缩酶、水化酶（脱水酶）、脱氨酶等。Lyases (class 4, often also referred to as“synthases”) catalyze reactions involving either the cleavage or formation of chemical bonds, with double bonds either arising or disappearing.(See figure- reversible reaction is shown). Cleavage of bond does not require water.

Isomerases Epimerases, cis-, trans- Isomerases, Intramolecular transferases 【异构酶类】催化各种同分异构体的相互转化，即底物分子内基团或原子的重排过程。 如磷酸己糖异构酶催化的反应。The isomerases (class 5) move groups within a molecule, without changing the gross composition of the substrate.  

Ligases / Synthetases C-C-Ligases, C-O-Ligases, C-N-Ligases, C-S-Ligases 【合成酶类】催化C-C、C-O、C-N 以及C-S 键的形成反应。这类反应必须与ATP分解反应偶联。例如谷氨酰胺合成酶催化的反应。The ligation reactions catalyzed by ligases (“synthetases,” class 6) are energy-dependent and are therefore always coupled to the hydrolysis of nucleoside triphosphates(See figur

eg. Lactate dehydrogenase Enzyme Commission (EC) EC 1. 1. 1. 27 Oxidoreductases Acting on the CH-OH group of donors. With NAD(+) or NADP(+) as acceptor. 【乳酸脱氢酶】[国际酶学委员会]; The EC numbers are organized in a four-level hierarchy, and each EC number consists of four numbers separated by dots. A hyphen (-) is used as a wild card, and numbers in the 90s are used to represent miscellaneous categories. A full, four-part EC number without any wild cards represents a specific enzyme. The example on the right shows the EC number for an enzyme that catalyzes reactions which transfer a specific type of phosphorous-containing sub-molecular group. In the Sprout Database, EC numbers are stored as part of a gene's functional assignment, as shown below. L-lactate dehydrogenase

Lecture 2 Kinetics of enzymatic reactions

What is enzymatic kinetics? Kinetics is the study of rates of chemical reactions Enzyme kinetics is the study of rates of chemical reactions that involve enzymes

The determination of enzymatic reaction velocity Effect of substrate concentration on enzymatic reaction velocity Effect of pH on enzymatic reaction velocity Effect of enzyme concentration on enzymatic reaction velocity Effect of temperature on enzymatic reaction velocity Effect of activators on enzymatic reaction velocity Effect of inhibitors on enzymatic reaction velocity 酶促反应速率的测定 底物浓度对酶作用的影响 pH对酶促反应速率的影响 酶浓度对酶促反应速率的影响 温度对酶作用的影响 激活剂对酶作用的影响 抑制剂对酶作用的影响

What is the velocity？ （反应初速度）

1. Measurement of enzymatic reaction velocity ΔP t Velocity —— The consumption of substrate or the formation of product per unit time v （反应速率）单位时间内底物的消耗量或产物的生成量。

Initial velocity It is given by the tangent at the origin to the curve of reaction velocity as a function of time. —— The reaction velocity in the earliest stage of an enzyme-catalysed reaction. In practice it is often measured over the period when the initial substrate concentration has diminished by <10%. （初速率）【切线】 底物浓度变化在5%以内的速率为反应初速率。

2. Effect of substrate concentrations on enzymatic reactions 一般反应,影响v的因素: 反应物浓度 t℃ P 介质pH 催化剂? 酶促反应 底物浓度 t℃ 介质pH 酶浓度 抑制剂 激活剂? Figure 6–11 Lehninger Principles of Biochemistry, 4/e©2006 D.L. Necson & M. M. Cox

中间产物学说解释只有少数的酶与底物作用生成中间产物，在这种情况下，增加底物的浓度，就会增加中间产物，从而增加酶促反应的速度 At relatively low concentrations of substrate, V0 increases almost linearly with an increase in [S].

At higher substrate concentrations, V0 increases by smaller and smaller amounts in response to increases in [S]. ES越多,反应速率取决于ES的浓度,反应速度随之增高

Finally, a point is reached beyond which increases in V0 are vanishingly small as [S] increases. This plateau-like V0 region is close to the maximum velocity, Vmax. 所有的酶都与底物结合生成中间产物，体系中已经没有游离态的酶了(酶全部被饱和)，继续增加底物的浓度，对于酶促反应的速度，显然已毫无作用

The Michaelis–Menten Equation 【米氏方程】

总酶浓度[E] 0，则[E] 0= [E] + [ES]

When [S] is high，all ES， V=Vmax

Michaelis constant（Km ） V0= Vmax[S] Km + [S] If v0＝Vmax/2 Km = [S] 1 V 2 ＝ V [S] Km + [S] 米氏常数 Km + [S] = 2[S]

V0 = 1/2 Vmax, Km =????? 当反应速度等于最大速度一半时,即，即米氏常数是反应速度为最大值的一半时的底物浓度。因此,米氏常数的单位

Km characteristic constant of enzyme 酶 底物 Km(mmol/L) 脲酶 尿素 25 溶菌酶 6-N-乙酰葡萄糖胺 0.006 6-磷酸葡萄糖脱氢酶 6-磷酸葡萄糖 0.058 胰凝乳蛋白酶 苯甲酰酪氨酰胺 2.5 甲酰酪氨酰胺 12.0 乙酰酪氨酰胺 32.0 （与酶及底物种类有关→鉴定酶）不同的酶具有不同Km值，它是酶特征物理常数，固定温度、pH、缓冲体系等条件。 Km is a characteristic constant of enzyme. Km is independent of [E]. It is determined by the structure of enzyme, the substrate and environmental conditions (pH, T, ionic strength, …)

Km may reflect the affinity of the enzyme for its substrate Km may reflect the affinity of the enzyme for its substrate. The larger the Km, the smaller the affinity. E + S ES P + E k1 k-1 k2 Km= k2 + k-1 k1 【亲和力】 Km大小近似表示酶对底物的亲和力大小（分离能力） Km≈k2（分离能力）/k1（亲和能力）

Lineweaver - Burke or double reciprocal plot 1 v = Km Vmax · [S] +

3. Effect of pH on enzymatic reaction velocity 胃蛋白酶胆碱酯酶提问：pH对酶活力产生影响的原因？答案：影响S和E的基团解离 → 影响二者的结合酶最适pH，因酶而异，大多数酶最适pH在7.0左右。 不是酶的特征常数 胰蛋白酶 过氧化氢酶精氨酸酶延胡索酸酶RNA酶

Enzymes have an optimum pH (or pH range) at which their activity is maximal. At higher or lower pH, activity decreases.

4. Effect of temperature on enzymatic reaction velocity The rates of enzyme-catalyzed reactions generally increase with increasing temperature. Thermal denaturation of protein structure at higher temperatures. 【热变性】

大部分酶60 ℃以上变性，少数例外，细胞淀粉酶90 ℃时活性很高。 At the optimum temperature, an enzyme has the maximal catalytic power. 温血动物体内酶最适温度：35—40℃ 植物体内酶最适温度：40—50 ℃ 微生物体内酶最适温度：40—50 ℃很高 大部分酶60 ℃以上变性，少数例外，细胞淀粉酶90 ℃时活性很高。 【最适温度】酶若制成干粉，可放置在室温下保存，而处于溶液状态时必须放在冰箱里保存。 提问：原因何在？ 答案：低温无水使微生物降解酶活性低

5. Effect of inhibitors on enzymatic reaction velocity —— any substance that inhibits an enzymatic reaction. 凡是能改变酶蛋白必需基团的化学性质而引起酶活力下降/丧失的作用为抑制作用 能够引起抑制作用的化合物称为抑制剂（inhibitor）。

Irreversible inhibition The irreversible inhibitors bind covalently with or destroy a functional group on an enzyme that is essential for the enzyme’s activity. （不可逆抑制作用）抑制剂与酶蛋白的必需基团以共价形式结合，引起酶的永久性失活。高浓度的重金属盐； 有机磷/汞/砷化合物、烷化剂（碘乙酸、碘乙酰胺）、酰化剂；有机磷化合物（抑制Ser-OH酶）有机磷化合物（抑制Ser-OH酶） 有机汞、有机砷化合物（抑制-SH硫基酶） 氰化物 (抑制含铁卟啉的酶 ) 烷化剂（碘乙酸、碘乙酰胺）（抑制-SH硫基酶） 氰化物、硫化物、CO

乙酰胆碱酯酶 （二异丙基氟磷酸）乙酰胆碱----传递神经冲动有机磷农药抑制胆碱酯酶活性 → 乙酰胆碱的堆积 → 神经过度兴奋 → 抽搐而死排毒 喝鸡蛋清、牛奶→洗胃 导泄、利尿 血液灌流（“大换血”） 血液透析（清除游离状态毒物） 找特效药（解磷定）

Heavy Metal Poisoning (Hg and Pb) -CH2-SH + Hg or Pb -CH2-S-Hg Pb Lead poisoning may have caused years of chronic illness in composer Ludwig van Beethoven and may have contributed to his death. Chemical analysis experts found unusually high levels of lead in strands of Beethoven's hair.

+ -CH2-OH -CH2-O-

Reversible inhibition —— A type of inhibition that occurs when the equilibrium between free inhibitor and the inhibitor - enzyme complex lies sufficiently far towards the free inhibitor state for the inhibitor to be readily removed, leaving an active enzyme. （可逆抑制作用）抑制剂与酶蛋白以非共价方式结合，引起酶活性暂时性丧失。 抑制剂可以通过透析等方法被除去，并且能部分或全部恢复酶的活性。

Competitive inhibition Reversible inhibition Noncompetitive inhibition Uncompetitive inhibition 不可逆抑制可逆抑制竞争性抑制非竞争性抑制 反竞争性抑制

Competitive inhibition Both the substrate and inhibitor competes with one another for the same active site resulting in competitive inhibition （竞争性抑制） I与S结构相似，能与S竞争与E活性中心结合。当I与活性中心结合后，S被排斥在反应中心之外，导致酶促反应被抑制了。

可以通过增大[S]来消除 生物碱麻醉----竞争性替代

动力学方程式及双倒数图

（1）Km增大 （2）Vmax不变

Treatment of HIV With AZT 葛兰素威康制药公司生产的一种抗艾滋病药物，据报道，它能够防止艾滋病病毒的母婴传播。 它又叫Zidovudine，商标名为Retrovir。 艾滋病病毒靠逆转录繁殖，而AZT是一种逆转录酶抑制剂，因为AZT的分子结构与胸腺嘧啶脱氧核苷酸的结构很相似。是评价其它抗HIV药物的阳性对照药，也是治疗HIV感染者和AIDS患者联合用药的基准药物。因而AZT被列为《国家级新药开发指南》第三辑推荐的抗病毒药物的首位．AZT对艾滋病毒有相当高的抑制力。对艾滋病的治疗开始阶段是单独使用，后与其它药物配合使用，是所有抗艾滋病药物中最具有价格优势的。目前世界年需求量为100吨，随着艾滋病患者的急剧增加，世界年需求量将逐渐增加至200吨，现市场上100吨AZT中有80%的市场份额被葛兰素公司占有。

Noncompetitive inhibition —— Inhibition of an enzyme in which inhibition results from binding of the inhibitor at a different site on the enzyme surface from that at which the substrate binds, without interference with substrate binding. （非竞争性抑制）S和I对E没有竞争作用。E与I结合后，还可与S结合；E与S结合后，也可再结合I，但是ESI三元中间产物不能进一步分解为产物，导致酶促反应被抑制。 如某些金属离子（Cu2+、Ag+、Hg2+）以及EDTA等，通常能与酶分子的调控部位中的-SH基团作用，改变酶的空间构象，引起非竞争性抑制。

动力学方程式及双倒数图

（1）Km不变，E与S亲和力不受影响 （2）Vmax减小，一部分E始终失活

Uncompetitive inhibition ——Uncompetitive inhibition, takes place when an enzyme inhibitor binds only to the complex formed between the enzyme and the substrate (the E-S complex).

EIS

Lecture 3 Mechanism of enzyme – catalyzed reactions

1. Factors of high catalytic efficiency Acid–base catalysis Covalent catalysis Proximity and orientation effects Metal Ion Catalysis

Acid-base catalysis Amino acid side chains that can donate or accept protons can participate in chemical reactions as acid or base catalysts

FIGURE 6–9 Amino acids in general acid-base catalysis. His 是酶的酸碱催化中最活泼的功能基团 FIGURE 6–9 Amino acids in general acid-base catalysis.

胰凝乳蛋白酶通过酸碱催化使肽键断裂

Covalent catalysis Nucleophilic groups can catalyze reactions through the transient formation of covalent bonds with the substrate. 【共价催化】 E+S→反应活性很高的共价过渡产物→反应活化能降低→提高反应速度 亲核基团：His 的咪唑基，Cys 的-SH，Asp 的-COOH，Ser 的-OH等； 某些辅酶（如TPP和PLP）也可参与共价催化作用。 (an enzyme with a nucleophilic group X:)

Proximity and orientation effect Enzymes bring their substrates together in proper spatial relationships for reactions to occur The substrate must come into close proximity to catalytic functional groups The substrate must be precisely, spatially oriented to the catalytic groups 【邻近与定向效应】 普通化学反应：随机碰撞（受浓度、碰撞角度影响）邻近作用缩短了酶催化基团与底物活性基团的距离 定向作用正确定位了底物与催化基团间的位置 提高反应速度（成功率）108倍

既有“邻近”又有“定向”

Metal Ion Catalysis Metals Most common cofactors - iron, copper, manganese and cobalt Structural ions - sodium, potassium and calcium Both - magnesium and zinc

2. Mechanism of high catalytic efficiency Enzyme-catalysis and activation energy Activated molecules → crash effectively → chemical reactions The reaction rate depend on the number of activated molecules. The ways that accelerate the rates of reactions： Increase in energy → increases the number of activated molecules； Catalysts → lower the energy of activation → increases the number of activated molecules 【活化能】

3. Intermediate theory E + S E-S  P + E 【中间产物】在酶促反应中，E与S首先形成E-S中间复合物。当S在E作用下发生化学变化后，E-S中间复合物再分解成E和P。

http://www. livingscience. co. uk/joomla/index. php http://www.livingscience.co.uk/joomla/index.php?option=com_content&view=article&id=57:enzymes&catid=42:enzymes&Itemid=54

Lecture 4 Regulation of enzyme activities

Regulating enzyme activity Modes of regulation: 1. Allosteric Control 2. Covalent Modification

1. Allosteric Regulation Some enzymes do not display the characteristics of Michaelis-Menten kinetics. These enzymes are called, allosteric or regulatory enzymes These enzymes are responsible not only for catalysis but also for regulating the overall rate of metabolic processes.

Allosteric enzymes Allosteric enzymes function through reversible, noncovalent binding of regulatory compounds called allosteric modulators or allosteric effectors, which are generally small metabolites or cofactors.

Regulation of allosteric enzymes by modulators T state R state

Allosteric enzymes can be used to regulate enzymatic pathways Allosteric enzymes can be used to regulate enzymatic pathways. In a negative-feedback pathway, the end product of a pathway binds to the allosteric site of the first enzyme, closing its active site and blocking the rest of the reactions.

The features of allosteric enzyme Enzyme activities are changed by metabolic activator or inhibitors. Allosteric effectors bind noncovalently to the enzymes they regulate. Multi-subunit proteins. Vo versus [S] curve shows sigmoid. [S型] [协同 ] 1）寡聚酶，某些酶0℃不稳定，室温稳定 活性中心——底物 2）酶分子 别构中心或调节中心——效应物 3）引起别构酶别构效应的物质称效应物(调节物） 正效应物——增加v（底物） 负效应物——降低v（终产物） 4）别构酶具有协同效应(cooperativity) 5）shows sigmoid [S型] kinetics

别构酶的动力学特征

2. Covalent Modification The enzyme activity can be modified by the covalent attachment and removal of groups on the polypeptide chain. Covalent modification of regulated enzymes must be reversible. The modifications usually require additional modifying enzymes for activation and inactivation. The most common type of covalent modification is phosphorylation.

Modifying groups are attached to an enzyme by a covalent bond.

Phosphorylation of enzymes by protein kinases Phosphoryl group is removed by protein phosphatases