Transcript K m + [S]

Chapter 3 Enzymes
• Almost all processes in the living cell are catalyzed
by the specific biocatalyst. Enzymes are catalysts
that change the rate of a reaction without being
changed themselves. Enzymes are highly specific and
their activity can be regulated..
•Biocatalyst: enzymes and ribozyme.
•One of the most important functions of proteins is
their role as catalysts. Until recently, all enzymes were
considered to be proteins. Several examples of
catalytic RNA molecules have now been vertified.
Living processes consist almost entirely of biochemical
reactions. Without catalysts these reactions would not
occur fast enough to sustain life.
• Enzymes bind to one or more ligands,
called substratee, and convert them into
one or more chemically modified products.
1
•
•
•
•
•
Composition of enzymes
Simple enzyme and conjugated enzyme.
Conjugated enzyme:
apoenzyme + cofactor
holoenzyme.
Cofactor : prosthetic group+ coenzyme
prosthetic group: tightly bond with apoenzyme.
FAD, metal, etc.
• coenzyme loosely bond with apoenzyme. NAD,
NADP, etc.
• Active site: Each type of enzyme molecule
contains a unique, intricately shaped
binding surface called an active site.
• Catalytic residues are highly conserved.
Certain amino acids, notably cysteine and
hydroxylic, acidic, or basic amino acids,
perform key roles in catalysis.
• Essential group in active site: binding
group +catalytic group. Cofactors always be
a part of the active site.
Active site
• The active site is the region of the enzyme that
binds the substrate, to form an enzyme-substrate
complex, and transforms it into product. The
active site is a three-dimensional entity, often a
cleft or crevice on the surface of the protein, in
which the substrate is bound by multiple weak
interactions. Two models have been proposed to
explain how an enzyme binds its substrate: the
lock-and –key model and the induced-fit model.
2
Characteristics and mechanisms
of enzymatic reactions
• Characteristics
• Enzymes have several remarkable properties. First,
the rates of enzymatically catalyzed reactions are
often phenomenally high. (Rate increases by factors of
106or greater are common.) . Second, in marked
contrast to inorganic catalysts, the enzymes are highly
specific to the reactions they catalyze. Side products
are rarely formed. Finally, because of their complex
structures, enzymes can be regulated. This is an
especially important consideration in living organisms,
which must conserve energy and raw materials.
• Specificity: Absolute specificity, relative specificity, and
stereospecificity.
• Activation energy: To proceed at a viable rate, most
chemical reactions require an initial input of energy. In
the laboratory this energy is usually supplied as heat. At
temperatures above absolute zero (-273.1ºC), all
molecules possess vibrational energy, which increases as
molecules are heated. Consider the following reaction:
A+B
C
As the temperature rises, vibrating molecules (A and B)
are more likely to collide, A chemical reaction occurs
when the colliding molecules possess a minimum amount
of energy called the activation energy.
Activation energy
Uncatalyzed
Energy
activation energy
Non-enzymatic
activation energy
Enzymatic
activation energy
Substrate
Total energy
Changes of reaction
Product
Progress of reaction
• Not all collisions result in chemical reactions
because only a fraction of the molecules have
sufficient energy.
• Induced-fit hypothesis and transition state.
Substrates induce conformational changes in
enzymes. During any chemical reaction reactants
with sufficient energy will attain transition state
(a strained intermediate form) when the substrate
binds to the enzyme (inducing).
Induced-fit Theory
substrate
Complex of substrate-enzyme
enzyme
• Mechanisms
Proximity effect and orientation arrange: For a
biochemical reaction to occur, the substrate must
come into close proximity to catalytic functional
groups (side chain groups involved in a catalytic
mechanism ) within the active site. In addition, the
substrate must be precisely, spatially oriented to
the catalytic groups. Once the substrate is
correctly positioned, a change in the enzyme’s
conformation may result in a strained enzymesubstrate complex. This strain helps to bring the
enzyme-substrate complex into the transition state.
• Multielement catalysis (Acid-Base catalysis ) :
Chemical groups can often be made more reactive
by adding or removing a proton. Enzyme active
sites contain side chain groups that act as proton
donors or acceptors. These groups are referred to
as general acids or general bases.
• Surface effect: The strength of electrostatic
interactions is related to the capacity of
surrounding solvent molecules to reduce the
attractive forces between chemical groups. Water
is largely excluded from the active site as the
substrate binds.
3
Enzyme kinetics
• The rate or velocity of a biochemical reaction is
defined as the change in the concentration of a
reactant or product per unit time.
• Plotting initial velocity v versus substrate
concentration [S].The rate of the reaction is directly
proportional (first order reaction) to substrate
concentration only when [S] is low. When [S]
becomes sufficiently high that the enzyme is
saturated, the rate of the reaction is zero-order with
respect to substrate.
V
Michaelis-Menten Equation
k1
S+E
k2
ES
k3
E+P
K1= rate constant for ES formation
K2= rate constant for ES dissociation
K3= rate constant for product formation
and release from the active site
(1)
v=
Vmax [S]
Km + [S]
(2)
k1
S+E
k2
ES
k3
E+P
ES formation = K1 ( [E] - [ES] ) [S]
(3)
ES dissociation = K2 [ES ]+ K3 [ES]
(4)
K1 ( [E] - [ES] ) [S] = K2 [ES ]+ K3 [ES]
( [E] - [ES] ) [S]
K2+ K3
=
[ES]
K1
Michaelis and Menton introduced a new constant,
Km ( now referred as the Michaelis constant):
K2+ K3
Km=
K1
( [E] - [ES] ) [S]
Km =
[ ES]
Km [ES] = [E] [S] – [ES] [S]
Km [ES] + [ES] [S] = [E] [S]
[ES] ( Km + [S] ) = [E] [S]
[E] [S]
[ES] =
(5)
Km+[S]
Since V= K3 [ES], from ( 5 )
[E] [S]
V= K3
(6)
Km+[S]
When the [S] is much higher than the enzymes, all
enzymes form [ES], that is, [E]= [ES], and maximum
velocity ( Vmax ) can attain.
Vmax = K3 [ES] = K3 [E]
Vmax
K3 =
[E]
(7)
Vmax [E] [S]
V=
Vmax [S]
=
[E]
Km+[S]
(2)
Km+[S]
Significances of Km and Vmax
1) When [S] = Km,
Vmax [S]
Vmax
V=
=
[S] + [S]
2
2) When [S] is very much greater than Km,
Vmax [S] Vmax [S]
V=
=
= Vmax
Km+[S]
[S]
3) It may reflect the affinity of the enzyme for its
substrate. If K3 is much smaller than K2, that is K3 « K2,
Km is the dissociation constant for the [ES].
K2
K m=
K1
4) From Vmax = K3 [ES] = K3 [E], enzymes are saturated.
Vmax
K3=
[E]
The turnover number (Kcat ) = K3. This quantity is the
number of moles of substrate converted to product each
second per mole of enzyme.
Lineweaver-Burk Double-reciprocal
plot
.
Km
1
1
1
+
v = V
Vmax
max [S]
y = mx + b
.
Km
1
1
1
+
v = V
Vmax
max [S]
Slope
(intercept on the vertical axis)
(intercept on the horizontal axis)
Multiple factors affect the rates of
enzyme-catalyzed reactions.
• Temperature
While raising temperature increases the rate of an
enzyme-catalyzed reaction, this holds only over a
strictly limited range of temperatures. The reaction
rate initially increases as temperature rises owing to
increased kinetic energy of the reacting molecules.
Eventually, however, the kinetic energy of the enzyme
exceeds the energy barrier for breaking the weak bonds
that maintain its secondary-tertiary structure. At this
temperature, denaturation, with an accompanying
precipitate loss of catalytic activity, predominates.
• Enzymes from humans, who maintain a body
temperature of 37 ºC, generally exhibit stability at
temperature up to 45-55 ºC. Enzymes from
microorganisms that inhabit natural hot springs or
hyperthermal vents on the ocean floor may be
stable at or above 100 ºC.
• Optimum temperature: Temperature at which it
operates at maximal efficiency.
Enzyme activity
Temperature
( °C )
• pH
When enzyme activity is measured at several pH
values, optimal activity typically is observed between
pH values of 5 and 9. However, a few enzymes are
active at pH values well outside this range.
pH optimum: The pH value at which an enzyme’s
activity is maximal is called the pH optimum.
• Initial rate is proportionate to enzyme
concentration
The initial rate of a reaction is the rate
measured before sufficient product has been
formed to permit the reverse reaction to occur. The
initial rate of an enzyme-catalyzed reaction is
always proportionate to the concentration of
enzyme. Note, however, that this is statement
holds only for initial rates.
• Substrate concentration
酶浓度对反应速度的影响
• 当[S]>>[E]时，
[E]与v呈正比
关系。
Enzyme activity
pH dependent of enzyme activities
Pepsin
Amylase
pH
Acetylcholinesterase
(4)
Enzyme inhibition
The activity of enzymes can be inhibited. Many substances
can reduce or eliminate the catalytic activity of specific
enzymes. Inhibition may be irreversible or reversible.
Irreversible inhibitors usually bond covalently to the
enzyme, often to a side chain group in the active site. For
example, enzymes containing free sulfhydryl groups can
react with alkylating agents such as iodoacetate and heavy
metals. This process is not readily reversed either by
removing the remainder of the free inhibitor or by
increasing substrate concentration.
Specific inhibitor: specifically bind to essential amino acid
on active site. Some organic phosphor compounds could
specifically bind to –OH of serine.
Non specific inhibitor: not only binds to essential
group, but also to outsides of essential group. Hg2+,
Ag2+ and As3+ .
In reversible inhibition:
the inhibitor can dissociate from the enzyme because it
binds through noncovalent bonds. The most common
forms of reversible inhibition are competitive and
noncompetitive.
1)
Competitive inhibition
• Competitive inhibitors typically resemble
the substrate
• Classic competitive inhibition occurs at
the substrate-binding (catalytic) site. The
chemical structure of a substrate analog
inhibitor (I) generally resembles that of the
substrate (S). It therefore combines
reversibly with the enzyme, forming an
enzyme-inhibitor (EnzI) complex rather than
an EnzS complex.
Competitive inhibition
I
Ki
v=
EI
E+P
ES
E+S
+
Vmax [S]
[I] )£)+ [S]
Km (1+
Ki
Km
[I] ) £)1
1
1
(1+
=
+
v
Vmax
Ki [S] Vmax
inhibitor
No inhibitor
Noncompetitive inhibition
• In noncompetitive inhibition, no competition
occurs between S and I. The inhibitor
usually bears little or no structural
resemblance to S and may be assumed to
bind to the enzyme at a site other than the
active site. Both EI and EIS complexes
form. Inhibitor binding alters the enzyme’s
three-dimensional configuration and blocks
the reaction.
Noncompetitive inhibition
E+S
+
I
Ki
EI + S
ES
+
I
Ki
ESI
E+P
Plots of 1/V versus 1/[S] in the
presence of several concentrations of the
inhibitor intersect at the same point on
the horizontal axis, -1/Km. In
noncompetitive inhibition the dissociation
constants for ES and EIS are assumed to
stay the same.
inhibitor
No inhibitor
3)
Uncompetitive inhibition
• The inhibitor bind to ES and results in
decrease of both ES and P (also free E).
• E+S
ES
E+S
+
I
Ki
ESI
Uncompetitive inhibition
E+S
ES
+
I
Ki
ESI
E+P

1 Km 1
1
I


(1  )
v Vmax S Vmax
Ki
inhibitor
No inhibitor
4) Effect of activator on the enzyme
activities
• Activator: substances enable non-active
enzyme to become active one. Metals such
as Mg2+, K+, Mn2+, etc.
• Essential activator and non-essential
activator.
5) Enzyme activity assay and unit of
enzyme activity
• Enzyme activity is measured in international units
(I.U.) One I.U. is defined as the amount of enzyme
that produces 1μmol of product per minute. An
enzyme specific activity, a quantity that is used to
monitor enzyme purification, is defined as the number
of international units per milligram of protein.
• A new unit for measuring enzyme activity called the
katal, has recently been introduced. One katal (kat)
indicates the amount of enzyme for the transformation
of 1 mole of substrate per second.
• 1 IU =16.67×10-9 kat
4 Regulation of enzyme
• The thousands of enzyme-catalyzed chemical
reactions in living cells are organized into a
series of biochemical or metabolic pathways.
Each pathway consists of a sequence of
catalytic steps. The product of the first
reaction becomes the substrate of the next
and so on. Metabolic and other processes are
controlled by altering the quantity or the
catalytic efficiency of enzymes.
1) Regulation of enzyme activities
• A. Proenyme or Zymogen: Certain proteins are
manufactured and secred in the form of inactive
precursor proteins known as proproteins. When the
proteins are enzymes, the proproteins are termed
proenzymes or zymogens. Conversion of a
proprotein to the mature protein involves selective
proteolysis, a process that converts the proprotein by
one or more successive proteolytic “clips” to a form
having the characteristic activity of the mature
protein ( its enzymatic activity ). Examples include
the hormone insulin (proinsulin), pepsinogen,
trypsinogen, etc.
• Selective proteolysis of a proenzyme may
be viewed as a process that triggers
essential conformational changes that
“create” the catalytic site.
B. Allosteric enzyme
•
Allosteric enzymes are enzymes whose
activity at the catalytic site may be modulated by
the presence of allosteric effectors at an allosteric
site. Allosteric effector could be products,
substrate, and so on.
• Feed back inhibition referred to the inhibition of
the activity of an enzyme in a biosynthetic
pathway by an end product (often as allosteric
effectors) of that pathway.
C. Regulatory covalent modification
• Regulatory covalent modifications can be
reversible or irreversible. In mammalian cells,
the two most commonly used forms of covalent
modification are partial proteolysis and
phosphorylation. Because cells lack the ability
to reunite the two portions of a protein
produced following hydrolysis of a peptide
bond, the partial proteolysis is considered an
irreversible modification.
• Hydrolysis of the phosphoesters formed
when a protein is covalently phosphorylated
on the side chain of a serine, threonine, or
tyrosine residues is both thermodynamically
spontaneous and readily catalyzed by
enzymes called protein phosphatases. Hence,
phosphorylation represents a reversible
modification process.
Cyclic phosphorylation
and dephosphorylation
is a common cellular
mechanism for
regulating protein
activity. In this example,
the target protein R
(orange) is inactive when
phosphorylated and
active when
dephosphorylated; the
opposite pattern occurs in
some proteins.
2) Regulation of enzyme quantity
• Rate of synthesis and degradation determine enzyme
quantity. The quantity of an enzyme in a cell may be
increased either by elevating its rate of synthesis, by
decreasing its rate of degradation, or by both. Cells can
synthesize specific enzymes in response to changing
metabolic needs, a process referred to as enzyme
induction. The induction accomplished by genetic
control. Although many inducers are substrates for the
enzymes they induce, compounds structurally similar to
the substrate may be inducers but not substrates.
Conversely, a compound may be a substrate but not an
inducer.
• The synthesis of certain enzymes may also be
specifically inhibited. In a process called repression,
the end product of a biochemical pathway may
inhibit the synthesis of a key enzyme in the pathway.
Both induction and repression involve cis-elements,
specific DNA sequences located upstream of genes
that encode a given enzyme, and a trans-acting
regulatory proteins.
• Regulation of enzyme degradation. The
degradation of mammalian proteins by ATP and
ubiqitin-dependent pathways and by ATPindependent pathways. It also Related to the
nutrition and hormone state.
Compartmentation
•
In eukaryotic cells, biochemical pathways are
segregated into different organelles. One purpose for
this physical separation is that opposing processes are
easier to control if the occur in different
compartments. For example, fatty acid biosynthesis
occurs in the cytoplasm, while the energy-generating
reactions of fatty acid oxidation occur within the
mitochondria. Another factor is that each organelle
can concentrate specific substances such as substrates
and coenzymes. In addition, special
microenvironments are often created within
organelles.
3) Isoenzymes
• The enzymes catalyzing the same
biochemical reaction.
• Lactate dehydrogenase (LDH)
Isoenzymes
H subunit
M subunit
Isoenzymes of lactate dehydrogenase
5 Nomenclature and classification
• The International Union of Biochemistry
(IUB) adopted a complex but unambiguous
system of enzyme nomenclature based on
reaction mechanism.
• (1) Reactions and the enzymes that
catalyzed them form six classes, each
having 4-13 subclasses.
• (2) The enzyme name has two parts. The first
names the substrate or substrates. The second,
ending in –ase, indicates the type of reaction
catalyzed.
• (3) Additional information, if needed to clarify the
reaction, may follow in parentheses; eg, the enzyme
catalyzing
• L-malate + NAD+
pyruvate + CO2 + NADH + H+
is designated 1.1.1.37 L-malate:
NAD+ oxidaoreductase (decarboxylating).
• (4) Each enzyme has a code number (EC) that
characterizes the reaction type as to class, subclass,
and subsubclass.
Classification
•
•
•
•
•
Six classes based on reaction mechanism:
(1) Oxidoreductases: LDH, Cytochrome C, etc.
(2) Transferases: methyl transferase.
(3) Hydrolases: amylase
(4) Lyases removing a group to form a double bond,
or reverse reaction.
• (5) Isomerase to catalyze the intertransfer of isomers.
• (6) Ligase. catalyzing two substrates link to form one
compound.
Relationship between Enzyme and
Medicine
选择题练习
酶化学
1.
关于酶概念的叙述下列哪项是正确的？
A．所有蛋白质都有酶的活性
B．其底物都是有机化合物
C．其催化活性都需要特异的辅助因子
D．体内所有具有催化活性的物质都是酶
E．酶是由活细胞合成具有催化作用的蛋白质
2．关于酶活性中心的叙述下列哪项是正确的？
A．所有酶的活性中心都有金属离子
B．所有的抑制剂都作用于酶的活性中心
C．所有的必需集团都位于酶的活性中心
D．所有酶的活性中心都含有辅酶
E．所有的酶都有活性中心
3. 酶加速化学反应的根本原因是(
A. 升高反应温度
B. 增加反应物碰撞频率
C. 降低催化反应的活化能
D. 增加底物浓度
E. 降低产物的自由能
)
4. Holoenzyme refer to (
)
A. Complex of enzyme with substrate
B. Complex of enzyme with suppressant
C. Complex of enzyme with cofactor
D. Inactive precursor of enzyme
E. Complex of enzyme with allosteric effector
5. 金属离子作为辅助因子的作用错误的是(
A. 作为酶活性中心的催化基团参加反应
B. 与稳定酶的分子构象无关
C. 可提高酶的催化活性
D. 降低反应中的静电排斥
E. 可与酶、底物形成复合物
)
6. 活化能的概念是指(
)
A. 底物和产物之间能量的差值
B. 参与反应的分子所需的总能量
C. 分子由一般状态变成活化态所需能量
D. 温度升高时产生的能量
E. 以上都不是
7. 酶促反应动力学所研究的是(
A. 酶的基因来源
B. 酶的电泳行为
C. 酶的诱导契合
D. 酶分子的空间结构
E. 影响酶促反应速度的因素
)
8. Michaelis-Menten enzyme kinetics
diagram of curves is a ( )
A. straight line
B. rectangular hyperbola
C. S shape curve
D. parabola
E. Not above all
9. 关于Km的意义正确的是(
)
A. Km为酶的比活性
B. 1/Km越小,酶与底物亲和力越大
C. Km的单位是mmol/min
D. Km值是酶的特征性常数之一
E. Km值与酶的浓度有关
10. 竞争性抑制剂的特点是(
)
A. 抑制剂以共价键与酶结合
B. 抑制剂的结构与底物不相似
C. 当抑制剂的浓度增加时,酶变性失活
D. 当底物浓度增加时,抑制作用不减弱
E. 抑制剂和酶活性中心外的部位结合
11. In anticompetitive inhibition of enzyme, the
reaction kinetics parameter change as ( )
A. Km↑,Vmax invariably
B. Km↓,Vmax↓
C. Km invariably,Vmax↓
D. Km↓,Vmax invariably
E. Km↓,Vmax↑
12. 有机磷农药与酶活性中心结合的基团是(
A. 组氨酸上的咪唑基
B. 赖氨酸上的ε-氨基
C. 丝氨酸上的羟基
D. 半胱氨酸上的巯基
E. 谷氨酸上的γ-羧基
)
13.
关于变构酶的论述错误的是(
)
A. 变构酶为多亚基组成
B. 如底物与一亚基结合后,使其他亚基迅速与底
物结合程正协同效应
C. 正协同效应的底物浓度曲线呈矩形双曲线
D. 底物与一亚基结合后,使其亚基结合底物能力
减少称负协同效应
E. 变构效应剂与一亚基结合后,使酶其他亚基迅
速与底物结合为异促协同效应
14.
–SH is one enzyme’s essential group. Which
substance can protect this enzyme from oxidation?
A. Cys
B. GSH
C. urea
D. ionic detergent
E. ethanol
15. 快速调节可通过(
)
A. 磷酸化与去磷酸化
B. 腺苷酸化与腺苷酸化
C. 变构调节
D. 改变酶的合成速度
E. 酶促反应的可调节性
16. The characteristic constants of
enzymes include ( )
A. Enzymic optimum temperature
B. Enzymic optimum pH
C. Vmax
D. Km
E. KS
17. 磺胺药的抑菌机理是(
)
A. 竞争性抑制二氢叶酸合成酶的活性
B. 干扰体内核酸的代谢
C. 结构与二氢叶酸相似
D. 抑制程度强弱取决于药物与酶底物浓度
的相对比例
E. 磺胺药是二氢叶酸合成酶的变构抑制剂
18. Cofactors of enzyme are (
)
A. Micromolecule organic compounds
B. metal ion
C. vitamine
D. various kinds of organic and
inorganic compounds
E. A kind of conjugated protein
19. 某种酶的活性依赖于酶活性中心的必需基团
-SH,能保护此酶不被氧化的物质是(
)
A. GSH
B. 维生素C
C. 半胱氨酸
D. 维生素A
E. 两价阳离子
20. 酶分子上必需基团的作用是(
A. 与底物结合
B. 催化底物发生化学反应
C. 含砷的有机化合物
D. 决定辅酶结构
E. 维持酶分子空间结构
)
Thank you!