生物化学sectionC

C1 Introduction to enzymes

s as catalysts酶作为催化剂

Enzymes are catalysts that change the rate of a chemical reaction without being changed

themselves in the process.

酶是一种催化剂，它可以改变化学反应的速率，而不会在反应过程中改变自身。

Four characteristics of enzymes:

(1)High efficiency（高效）

(2)Mild conditions （温和）temperatures below 100℃, atmospheric pressure and neutral pH.

温度低于100℃，大气压和中性pH。

(3) High specificity （专一）

There are three types of specificities.

te specificity（绝对专一）:

Enzymes can catalyze only one reaction.酶只能催化一个反应。

The activation of amino acids in translation needs the absolute specificity of aminoacyl tRNA

synthetase

翻译中氨基酸的激活需要氨基酰基tRNA合成酶的绝对特异性

ve specificity （相对专一）:

Enzymes catalyze one class of substrates or one kind of chemical bond in the same type.

酶催化一类底物或一种化学键。

specificity （立体异构专一）:

The enzyme can act on only one form of pisomers of the substrates.

这种酶只能作用于底物的一种同分异构体。

(4) enzyme activity can be regulated（可调）: in response to the concentration of substrates or

other molecules.

酶活性可以被调节:响应底物或其他分子的浓度

site活性位点

is the region that binds the substrate and converts it into product.

是与底物结合并将其转化为产物的区域。

The substrate(s) is bound in the active site by weak forces and reversible covalent bonds.

底物和活性位点结合通过可逆共价键和弱力（静电相互作用力、氢键、范德华键、疏水相互

作用力）

定义：指必需基团在空间结构上彼此靠近，组成具有特定空间结构的区域，能与底物特异结

合并将底物转化为产物

必需基团：酶分子中氨基酸残基侧链的化学基团中，一些与酶活性密切相关的化学基团。

活性中心内的必需基团：结合基团、催化基团

活性中心外的必需基团：位于活性中心以外，维持酶活性中心应有的空间构象和（或）作为

调节剂的结合部位所必需。

The model of enzyme binds its substrate:

lock-and-key model锁钥匙模型

The two shapes are considered as rigid and fixed这两种形状被认为是刚性的和固定的

induced-fit model诱导契合模型

The reality is that different enzymes show features of both models, with some complementarity

and some conformational change.

现实是，不同的酶表现出两种模型的特征，具有一定的互补性和一定的构象变化。

ate specificity 底物特异性

• The properties and spatial arrangement of the amino acid residues forming the active site of

an enzyme will determine which molecules can be substrates.

• Substrate specificity is often determined by changes in relatively few amino acids in the active

site.

形成酶活性位点的氨基酸残基的性质和空间排列将决定哪些分子可以是底物。

底物的特异性通常是由活性位点上相对较少的氨基酸的变化所决定的。

classification酶的分类

assays酶测定

The activity (活性) of an enzyme can be determined (assayed) in terms of the catalytic effect it

produces: the conversion of substrate to product.

一种酶的活性（活性）可以根据其产生的催化作用来确定：底物转化为产物。

An enzyme is assayed by measuring the rate of appearance of product or the rate of

disappearance of substrate.

一种酶是通过测量产物的出现速率或底物的消失速率来测定的。

Before we assay an enzyme,we need to know:

• the overall equation of the reaction；

• an analytical procedure must be available；

• Optimal working environment including cofactor, pH, temperature, etc.;

• Sufficient supply of substrate.

反应的整体方程；

必须有一个分析程序；

最佳的工作环境，包括辅助因子、pH、温度等。；

有足够的底物供应。

There are two methods used in the measurement of the enzyme activity.

1) Direct method:

If the substrate (or product) absorbs light, then changes of concentration can be measured by the

change of absorbance (吸光度).

如果基物（或产品）吸收光，则浓度的变化可以通过吸光度（吸光度）的变化来测量。

2) Indirect method: Linked enzyme assays

Reactions do not involve substrates or products that absorb light or fluoresce.

反应不涉及吸收光或荧光的基质或产物。

To assay the enzyme by linking it to a second enzyme reaction that involve an absorbance

change.

通过将该酶与涉及吸光度变化的第二个酶反应连接起来来分析该酶。

葡萄糖氧化酶的作用不会导致底物转换为产物时吸光度的变化。然而，产生的过氧化氢可被

过氧化氢酶作用，同时将无色化合物转化为有色化合物，其吸光度易测量。

5. Coenzymes and prosthetic groups辅酶和辅基

Simple enzymes (单纯酶):

consists of only one peptide chain

只包含一个肽链

Conjugated enzymes (结合酶):

holoenzyme = apoenzyme + cofactor

全酶=脱酶+辅助因子

酶蛋白和辅酶单独存在时均无催化活性，只有全酶才具有催化作用。

全酶分子中各部分在催化反应中的作用:

酶蛋白决定反应的特异性及其催化机制

辅助因子决定反应的性质与类型

Two types of cofactors:

1. metal ions

2. small organic molecules (coenzyme)

1. 金属离子2.小的有机分子（辅酶）

辅助因子按其与酶蛋白结合的紧密程度与作用特点不同可分为辅酶和辅基

辅酶多通过非共价键与酶蛋白结合，这种结合比较松散。

辅基通过共价键与酶蛋白结合，结合比较紧密。

A cofactor that is covalently attached to the enzyme is called a prosthetic group (辅基).

一种共价连接在酶上的辅助因子被称为辅基（辅基）。

(1) Use metal ions as cofactors

• Metal-activated enzyme: ions loosely bound.

• Metalloenzymes: ions tightly bound.

金属活化酶：松散结合的离子。

金属酶：离子紧密结合。

(2) Using small organic molecules as cofactors以小的有机分子为辅助因子

• Many coenzymes are derived from vitaminssuch as VB1, VB2, VB6, VB12, VC, Folic acid (叶酸),

Niacin (尼克酸)

许多辅酶来源于维生素，如VB1、VB2、VB6、VB12、VC、叶酸（叶酸)、烟酸(尼克酸）

The structures of NADH and NADPH NADH和NADPH的结构

NAD+ is more commonly used in catabolic(breakdown) reactions,whilst NADP+ is used in the

anabolic(biosynthetic) reactions.

NAD+更常用于分解代谢（分解）反应，而NADP+用于合成代谢（生物合成）反应。

反应还涉及质子的转移。

There are two types of coenzymes of dehydrogenase:脱氢酶有两种：

1) NAD+, FAD, FMN are used in catabolic (break-down) reactions.

NAD+、FAD、FMN被用于分解代谢（分解）反应。

NADH, FADH2— Provide energy.

2) NADP+ is used in anabolic (biosynthetic) reactions.

NADP+用于合成代谢（生物合成）反应。

NADPH —— Provides H+.

ymes同工酶

Isoenzymes (isozymes) are different forms of an enzyme that catalyze the same reaction, but

exhibit different physical or kinetic properties, such as isolelectric point, pH optimum, substrate

affinity or effect of inhibitors.

同工酶（同工酶）是催化相同反应的酶的不同形式，但表现出不同的物理或动力学性质，如

等电点、pH最佳、底物亲和力或抑制剂的作用。

LDH isoenzymes have different catalytic activities

LDH（乳酸脱氢酶）同工酶具有不同的催化活性

LDH1 (H4) in heart muscle converts lactate to pyruvate (丙酮酸), and then to acetyl CoA (乙酰

CoA).

LDH5 (M4) in skeletal muscle converts pyruvate to lactate.

心肌中的LDH1(H4)将乳酸转化为丙酮酸（丙酮酸），然后转化为乙酰辅酶A(乙酰CoA)。

骨骼肌中的LDH5(M4)可将丙酮酸转化为乳酸。

H4，H3M主要在心脏和红细胞中发现

H2M2主要在大脑中发现

HM3，M4主要在肝脏和骨骼肌中

根据国际生化学会的建议，同工酶是由不同基因或复等位基因编码，或由同一基因转录生成

的不同mRNA所翻译的不同产物（一系列酶）组成的蛋白质。

同工酶存在于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，它使不同的

组织、器官和不同的亚细胞结构具有不同的代谢特征。这为同工酶用来诊断不同器官的疾病

提供了理论依据。

C2 thermodynamics热力学

Learning Objectives:

1. Understand the concepts of thermodynamics;

2. Understand the difference between ΔG‡ (活化能) and ΔG (自由

能) .

1.理解热力学的概念。

2.了解ΔG（活化能）和ΔG（自由能）之间的区别。

1. Thermodynamics热力学

Thermodynamics is the description of the relationships among the various forms of energy and

how energy affects matter on the macroscopic level.

As it applies to biochemistry, thermodynamics is most often concerned with describing the

conditions under which processes occur spontaneously.

热力学是描述各种形式的能量之间的关系，以及能量如何在宏观水平上影响物质。

由于它适用于生物化学，热力学最常关注的是描述过程自发发生的条件。

The first law of thermodynamics

• States that the total energy of a system and its surroundings is a constant.

• The change in energy of a system depends only on the initial and final states and not on how it

reached that state

说明一个系统及其周围环境的总能量是一个常数。

一个系统的能量的变化只取决于初始状态和最终状态，而不取决于它如何达到该状态

The second law of thermodynamics

• States that a process can occur spontaneously only if the sum of the entropies of the system

and its surroundings increases.

• However, the entropy changes are not readily measured

说明一个过程只有在系统及其周围环境的熵之和增加时才能自发地发生。

然而，熵的变化并不容易测量

• Proposed by Gibbs which combines the first and second laws of thermodynamics:

由吉布斯提出的，它结合了热力学第一定律和第二定律：

2 . Activation energy and transition state活化能和过渡态

(1)Transition state is an unstable chemical form part-way between the substrates and the

products.

过渡态是基质和产物之间的不稳定化学形式

(2) The Gibbs free energy of activation (△G‡) is equal to the difference in free energy between

the transition state and the substrate.

吉布斯活化自由能(△G活化能)等于过渡态与衬底之间的自由能之差。

3. Free energy change自由能变化

A negative △G indicates that the reaction is thermodynamically favorable in the direction

indicated.

△G为负，表明反应在指示方向上热力学有利。

酶通过促进底物形成 酶通过促进底物形成过渡态而提高反应速率

（一）酶比一般催化剂更有效地降低反应活化能

（二）酶-底物复合物的形成有利于底物转变成过渡态

1.诱导契合作用使酶与底物密切结合

酶与底物相互接近时，其结构相互诱导、相互变形和相互适应，进而相互结合。这一过程

称为酶-底物结合的诱导契合(induced-fit) 。

2.邻近效应与定向排列使诸底物正确定位于酶的活性中心

酶在反应中将诸底物结合到酶的活性中心，使它们相互接近并形成有利于反应的正确定向关

系。这种邻近效应(proximity effect)与定向排列(orientation arrange)实际上是将分子间的反应

变成类似于分子内的反应，从而提高反应速率。

3.表面效应使底物分子去溶剂化

酶的活性中心多是酶分子内部的疏水“口袋”，酶反应在此疏水环境中进行，使底物分子脱

溶剂化(desolvation)，排除周围大量水分子对酶和底物分子中功能基团的干扰性吸引和排斥，

防止水化膜的形成，利于底物与酶分子的密切接触和结合。这种现象称为表面效应(surface

effect)

4. Chemical equilibria化学平衡

At equilibrium the ratio of the concentrations of the substrate and product remains at a constant

value, the equilibrium constant (K) K is also given by the ratio of the forwardreaction rate (kf) and

the reverse reaction rate (kb).

在平衡时，底物和产物的浓度比值保持在一个恒定值，平衡常数(K)K也由正向反应速率(kf)

和反向反应速率(kb)之比给出。

K= kf/kb=[B]eq/[A] eq

Enzymes accelerate the attainment of the equilibrium position but do not shift its position.

酶加速了平衡位置的达到，但不改变其位置。

C3 Enzyme kinetics 酶动力学

1. Enzyme velocity酶速度

Definition:The rate of an enzyme-catalyzed reaction is often called its velocity.

定义：酶催化反应的速率通常被称为其速度。

Enzyme velocities are normally reported as values at time zero (initial velocity)

酶速度通常报告为零时刻（初始值速度）

Enzyme units酶单位

(1) enzyme unit (U)：amount of enzyme which will catalyze the transformation of 1μmol of

substrate per minute at 25℃ under optimal conditions for that enzyme.

酶单位(U)：在最佳条件下每分钟催化底物1μmol转化的酶量。

(2) katal：catalytic activity which will raise the rate of a reaction by one mole per second in a

specified system.

katal：在特定系统中，将反应速率提高一摩尔每秒的催化活性

(3) 1μmol/min = 1 U = 16.67 nanokat

activity (or total activity) ：the total units of enzyme in the sample.

活性（或总活性）：样品中酶的总单位。

specific activity：the number of enzyme units per milligram of protein (units mg-1).It is a measure

of the purity of an enzyme.

比活性：每毫克蛋白质酶单位（单位毫克1）。它是一种衡量一种酶的纯度的方法。

2. Substrate and enzyme concentration底物和酶的浓度

substrate concentration

当底物浓度较低时，反应速度与底物浓度成正比；反应为一级反应

随着底物浓度的增高反应速度不再成正比例加速；反应为混合级反应。

当底物浓度高达极大时反应速度不再增加，达最大速度，反应为零级反应

Q1: Why the effect of substrate concentration appear a hyperbolic curve?

An enzyme-substrate complex, a transition state, is a key step in the catalytic reaction.

酶

-

底物复合物，一种过渡态，是催化反应的关键步骤。

Enzyme concentration

A doubling of the enzyme concentration will lead to a doubling of V0.

酶浓度增加一倍就会导致v0增加一倍。

3. Temperature

温度会以两种方式影响酶催化反应的速率。首先，温度升高提高了具有足够能量克服吉布斯

自由能的底物分子的比例。第二，较高的温度会导致酶的变形。温度升高对酶反应速率的总

体影响是这两种相反影响之间的平衡。

酶的最适温度不是酶的特征性常数，它与反应时间有关。

4. pH

Every enzyme has an optimum PH

PH值与最佳值的微小偏差导致活性降低。由于许多维持其三维结构的弱共价键受到干扰，

酶蛋白自身发生很大的变异，从而导致变性。

不同酶的最适PH值存在很大的差别，因为它们在不同的环境下工作。

Q2: Why the optimal pH of pepsin and trypsin is acidic and basic, respectively?

Q2

：为什么胃蛋白酶和胰蛋白酶的最佳

pH

分别是酸性的和碱性的？

5. Michaelis-Menten model米氏模型

米氏模型使用以上催化概念

Michaelis-Menten equation米氏方程：:

Km, the Michaelis constant Km，米氏常数

The significance of Km:

(1) a measure of the affinity :A high Km indicates weak substrate binding, a low Km indicates

strong substrate binding

(2) its value is equivalent to the substrate concentration at which the velocity is equal to half of

Vmax.

（1）亲和的测量。高Km表明底物结合弱，低Km表明底物结合强

（2）其值等于速度等于Vmax一半的底物浓度。

Q3: What are the limitations of Michaelis-Menten model?

问题3：米氏模型的局限性是什么？

It is impossible to estimate Vmax (and hence Km) from a hyperbolic plot.

无法估计Vmax（因此Km）从双曲线图。

6. Lineweaver-Burk plot

Applications of Lineweaver-Burk plot:

(1) to determine Vmax and Km values

(2) to determine the types of an inhibitor

（1）来确定Vmax和Km值

（2）来确定抑制剂的类型

C4 Enzyme inhibition

inhibition酶抑制剂

Any molecule that acts directly on an enzyme to lower its catalytic rate is called an inhibitor.

任何直接作用于酶以降低其催化速率的分子都被称为抑制剂。

There are two types of inhibitors:

(1) normal body metabolites:Feedback regulation

正常机体代谢产物:反馈调节

(2) foreign substances:drugs; toxins

异物：药物；毒素

Two types of enzyme inhibition:

(1) irreversible不可逆

(2) Reversible可逆

1) competitive竞争

2) noncompetitive非竞争

3) uncompetitive无竞争

rsible inhibition不可逆抑制剂

Inhibitors which bind irreversibly to an enzyme often form a covalent bond to an amino acid

residue at or near the active site, and permanently inactivate the enzyme

不可逆地与酶结合的抑制剂通常与活性部位或其附近的氨基酸残基形成共价键，并使酶永久

失活

Susceptible amino acid residues include Ser residue which has reactive-OH (hydroxy) group

易感氨基酸残基包括具有活性羟基（羟基）的丝氨酸残基

organophosphorus poisoning有机磷中毒

Susceptible amino acid residues include Cysresidue which has reactive–SH (sulfhydryl) group

易感氨基酸残基包括具有活性sh（巯基）的半胱氨酸残基

Hg2+、Ag+、Pb2+、As2+ heavy metal poisoning

Hg2+、Ag+、Pb2+、As2+重金属中毒

3. Reversible competitive inhibition可逆的竞争抑制剂

Characteristics:

(1) has close structural similarities to the normal substrate.

与正常底物具有密切的结构相似性。

(2) At high substrate concentrations the action of a competitive inhibitor is overcome.

在高底物浓度下，竞争性抑制剂的作用被克服。

(3) There is no change in the Vmax but a increase in Km.

Vmax没有变化，但Km有所增加。

Example:

1. 丙二酸对琥珀酸脱氢酶的影响

2. 磺胺类药物的抑菌机制

4. Reversible noncompetitive inhibition可逆的非竞争性抑制剂

Characteristics:

(1) binds at a site other than the active site.

结合在活性位点以外的一个位点。

(2) cannot be overcome by increasing the substrate concentration.

不能通过增加底物浓度来克服。

(3) There is no change in the Km but a decrease in Vmax.

Km没有变化，但Vmax有下降。

5. Reversible uncompetitive inhibition可逆的反竞争性抑制剂

Characteristics:

(1) binds at a site of enzyme-substrate complexes .

结合在酶-底物复合物的一个位点。

(2) reducing both Vmax and Km.

同时降低了Vmax和Km。

C5 Regulation of enzyme activity酶活性的调节

ck regulation反馈调节

A common theme in the control of metabolic pathways is when an enzyme early on in the

pathway is inhibited by an end-product of the metabolic pathway in which it is is

called feedback inhibition and other takes place at the committed step in the pathway(conversion

of A to B)

代谢途径控制中的一个共同主题是，当途径早期的一种酶被它所参与的代谢途径的终产物所

抑制时。这被称为反馈抑制，通常发生在途径中的关键步骤（A转化为B）

Many metabolic pathways are branched.许多代谢途径都是有分支的。

A process of sequential feedback inhibition may operate where the end-product of one branch of

a pathway will inhibit the first enzyme after the branch point.

顺序反馈抑制过程中，一个分支的最终产物将抑制分支点后的第一个酶。

eric enzymes变构酶

A plot of V0 against [S] gives a sigmoidal curve.

V0与[S]的曲线图给出了一条S形曲线。

Particularly sensitive to small changes in substrate concentration within the physiological range.

对生理范围内底物浓度的微小变化特别敏感。

The binding of a substrate molecule to one active site affects the binding of substrate

molecules to other active sites in the enzyme.

底物分子与一个活性位点的结合会影响底物分子与酶中其他活性位点的结合。

Two models to explain the allosteric effects observed in proteins.

在对称和协同模型中，变构酶的亚单位只能以两种状态之一存在，既T或R。

T状态亚单位处于紧致且相对不活跃的紧张状态。R状态亚单位处于放松、活性状态，对底

物亲和力更高。不允许中间状态。

在没有底物结合状态下，平衡有利于T状态。当底物以T状态结合到每个活性位点时，平衡

向R状态移动。所有亚单位以协调一致的方向改变构象。

在替代顺序模型中，当单个活性位点被占据后，寡聚酶的结构发生顺序变化。

底物与一个位点的结合影响相邻活性位点的底物亲和力，而不一定诱导围绕整个酶的转变，

因此整个蛋白质的分子对称性不一定时保守的。

An allosteric activator increases the rate of enzyme activity.

An allosteric inhibitor decreases the activity of the enzyme.

变构激活剂可以增加酶活性的速率。

变构抑制剂会降低酶的活性。

Aspartate transcarbamoylase (ATCase,天冬氨酸转氨基甲酰酶) is a key enzyme in pyrimidine

biosynthesis.

天冬氨酸转氨基甲酰化酶(ATCase，天冬氨酸转氨基甲酰酶)是嘧啶生物合成的关键酶。

3. Reversible covalent modification可逆共价修饰

is the making and breaking of a covalent bond between a nonprotein group and an enzyme

molecule.

是指非蛋白质基团和酶分子之间的共价键的形成和断裂。

The most common modification:

the addition and removal of a phosphate group (phosphorylation and dephosphorylation).

A phosphorylated enzyme may be either more or less active than its dephosphorylated form.

最常见的修饰是：添加和去除磷酸基（磷酸化和去磷酸化）。

磷酸化的酶活性可能比其去磷酸化的形式更多或更少。因此，磷酸化、去磷酸化可作为一种

快速、可逆的开关，根据需要打卡或关闭代谢途径。

蛋白激酶催化磷酸化，经常使用ATP作为磷酸供体

磷蛋白磷酸酶催化去磷酸化，以再生氨基酸中的羟基并且释放P

磷酸基团的添加或去除导致酶的三级结构发生变化，从而改变其催化活性。

The significance of reversible covalent

(1) Two forms ( higher or lower activity) easy for rapid regulation.

两种形式(活性高或低活性）易于快速调节。

(2) Amplification effect.A reaction chain consisted of several enzymes.

扩增效应。一个反应链由多种酶组成。

(3) consume one molecule of ATP. synthesis of enzyme (protein) needs more ATPs.

经济。只消耗一个ATP分子。酶（蛋白质）的合成需要更多的atp。

lytic activation酶原激活

(1) The inactive precursor form of enzyme called proenzyme or zymogen.

酶的非活性前体形式称为原酶或酶原。

(2) Activation of zymogens involves irreversible hydrolysis (水解) of one or more peptide bonds.

酶原的激活涉及一个或多个肽键的不可逆水解(水解）。

The significance of proenzyme activation酶原激活的意义

(1) to avoid self-digestion

e.g. Pancreatic proteases

(2) to activate at certain conditions

e.g. Blood clotting cascade

(3) to accumulate enzymes

(1)以避免自我消化，如胰腺蛋白酶(2)在某些条件下激活，如凝血级联(3）以积累酶

tion of enzyme synthesis and breakdown酶的合成和分解的调

控

Factors affecting the rate of synthesis include:

影响合成速率的因素包括

(1) the level of induction or repression of the gene基因的诱导或抑制水平

(2) rate of degradation of the mRNA mRNA降解速率

The rate of degradation of an enzyme is reflected in its half-life

一种酶的降解速率反映在其半衰期中