Download Rate Law in Enzyme Catalyzed Reactions

教
案
~ 2007 学年 第一 学期
2006
学 院
教
名 称
研
生命科学学院
室
课 程
名 称
授 课
对 象
授 课
教 师
陈文利
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副教授
职
教 材
名 称
2006 年 9 月
生物化学
2005 级生物技术专业
现代生物化学
日
授课题目（教学章、节或主题）
：
教学器材
与工具
第三章 酶 维生素与辅酶
授课时间
多媒体设施、黑板与
笔
第 6，7 周一第 15-20
节
教学目的、要求（例如识记、理解、简单应用、综合应用等层次）
：
主要掌握酶的概 念 , 六 大 分 类 ， 酶 的 单 底 物 动 力 学 曲 线 及 其 影 响 酶 活
力 的 因 素 重点掌握米式方程的计算及酶活力的定义方法，了解与代谢密切的辅酶结构特点，
了解与代谢密切的辅酶的主要功能
教学内容（包括基本内容、重点、难点）
：
Lecture Outline
What do enzymes do?
Why study enzymes? (Medical Relevance)
How do enzymes work?
What is the difference between an enzyme and a protein?
All enzymes are proteins except some RNAs and not all proteins are enzymes Enzymes
catalyze the conversion of substrates into products
What is a substrate?
–
A substrate is the compound that is converted into the product in an enzyme catalyzed
reaction.
–
For the reaction catalyzed by aldolase, fructose 1,6-phosphate is the substrate.
What is the difference between enzyme catalyzed reactions and uncatalyzed chemical
reactions?
Enzyme catalyzed reactions are much faster than uncatalyzed reactions.
Enzyme catalyzed reactions display saturation kinetics with respect to substrate concentration.
Enzyme catalyzed reactions are optimized for specific temperature and pH values.
Enzyme catalyzed reactions are much faster than uncatalyzed reactions
Enzyme catalyzed reactions display saturation kinetics with respect to reactant concentration
Enzyme Nomenclature
Oxidoreductases
(EC Class 1)
– Transfer electrons (RedOx reactions)
Transferases
(EC Class 2)
– Transfer functional groups between molecules
Hydrolases
(EC Class 3)
– Break bonds by adding H2O
Lyases
(EC Class 4)
– Elimination reactions to form double bonds
Isomerases
(EC Class 5)
– Intramolecular rearangements
Ligases
(EC Class 6)
– Join molecules with new bonds
Oxidoreductases catalyze the transfer of hydrogen atoms and electrons
Example - Lactate Dehydrogenase
Transferases catalyze the transfer of functional groups from donors to acceptors
Example - Alanine aminotransferase
Hydrolases catalyze the cleavage of bonds by the addition of water (hydrolysis)
Example - Trypsin
Lyases catalyze the cleavage of C-C, C-O, or C-N bonds
(addition of groups to double bonds or formation of double bonds by removal of groups)
Example - ATP-citrate lyase
Isomerases catalyze the transfer of functional groups within the same molecule
Example - Phosphoglucose isomerase
Ligases use ATP to catalyze the formation of new covalent bonds
Example - DNA ligase
Important things to remember about enzymes
1. Enzymes are not consumed or altered by the reaction they catalyze.
2. Enzymes catalyze both the forward and the reverse reaction.
Enzymes catalyze both the reactions in both the forward and reverse direction
3. Enzymes do not alter the equilibrium (or equilibrium constant) between substrates and
products.
How do enzymes work?
An energetic analysis.
Enzymes bring substrates together in the proper orientation for a reaction to occur
Enzymes possess functional groups that stabilize the transition state of the reaction
– Enzymes lower the activation Gibbs energy of a reaction
Enzymes drive thermodynamically unfavorable reactions
thermodynamically favorable reactions
by
coupling
them
to
Enzymes catalyze reactions by lowering their activation energies
Enzymes stabilize the transition states of reactions
The height of the transition state is a measure of the probability that the substrates will react
when they come in contact with each other
By lowering the height of the transition state, enzymes increase the probability that the
substrates will react when they come in contact with each other.
How do enzymes work?
The physical picture
Enzymes bind substrates to their active site and stabilize the transition state of the reaction.
What is the active site?
The active site of an enzyme is the place where all of the action occurs. It contains the
functional groups (amino acid side chains) that bind the substrate(s) and catalyze it’s
conversion to product(s).
Two models have been proposed for substrate substrate binding:
(1) Lock and key model
(2) Induced fit model
Emil Ficher: (1894)
–
The ‘Lock & Key’ hypothesis
- explains substrate specificity
- says nothing about why catalysis occurs
Dan Koshland: (1958)
–
‘Induced Fit’ hypothesis:
- enzymes prefer to bind to a distortion of the substrate that resembles the transition state
- both enzyme and substrate must adjust to one another
- in reality, enzyme is not ‘distorted’ but has evolved to bind in a certain way and
sometimes undergo conformational changes
Introduction to Enzyme Kinetics
Kinetics concerns with the rates of chemical reaction. Enzyme kinetics addresses the biological
roles of enzymatic catalysts and quantify the remarkable function of biological enzymes;
Enzyme kinetics information can be exploited to control and manipulate the course of metabolic
events. Pharmaceuticals or drugs are often special inhibitors targeted at a particular enzyme.
Thus the science of pharmacology relies on such information
Initial Velocity Assumption
In the beginning of the reaction, there is very little product, or [P] is small. So the amount of [ES]
contributed by E+P is negligible.
Thus, the MM equation concerns the reaction rate that is measured during early reaction
period.
In which case, the enzyme catalyzed reaction can be modified to:
Rate Law in Enzyme Catalyzed Reactions
Rate law still applies in enzyme catalyzed reactions.
The forward velocity, or rate, vf is,
The reverse velocity or rate, or the rate of disappearance vd is,
At steady state, there is no accumulation of [ES], thus:
Derivation of Michaelis-Menten Equation
We need one more condition, that is, the total enzyme concentration, [Et] is the sum of that of
enzyme-substrate complex, [ES], and that of free enzyme, [E]:
At steady state, the forward rate should equal to the reverse rate:
Rate of production formation (rate law), v = k2[ES]. So:
Notes on the MM Equations
The rate of production formation can usually be measured experimentally by monitoring the
progress curve of production formation.
The maximum rate can be reached at saturating substrate concentration, or when [S]
So MM equation can be re-written as:
Understanding Km
Km is a constant derived from rate constants
Km is, under true Michaelis-Menten conditions, an estimate of the dissociation constant of E
from S, because at equilibrium,
Reversible reaction, dissociation constant is
So small Km means tight substrate binding; high Km means weak substrate binding.
Km equals to the substrate concentration at which v=1/2vmax
Understanding Vmax
The theoretical maximal velocity
Vmax is a constant
Vmax is the theoretical maximal rate of the reaction - but it is NEVER achieved in reality
To reach Vmax would require that ALL enzyme molecules are tightly bound with substrate
Vmax is asymptotically approached as substrate is increased
The dual nature of the Michaelis-Menten equation
Combination of 0-order and 1st-order kinetics
When S is low, the equation for rate is 1st order in S
When S is high, the equation for rate is 0-order in S
The Michaelis-Menten equation describes a rectangular hyperbolic dependence of v on S!
Enzyme Inhibition
Enzyme can be inhibited by inhibitors. Inhibitors are tools to scientists to understand enzymes.
Inhibitors are also in many cases pharmaceutical reagents against diseases;
Inhibitors inhibit enzyme function by binding with enzymes. The binding reaction can be either
reversible or irreversible;
Reversible inhibitors associate with enzymes through non-covalent interactions. Reversible
inhibitors include three kinds:
Competitive inhibitors;
Non-competitive inhibitors;
Un-competitive inhibitors
Irreversible inhibitors associate with enzymes through covalent interactions. Thus the
consequences of irreversible inhibitors is to decrease in the concentration of active enzymes.
Summary of Classes of Inhibitors
Competitive inhibition - inhibitor (I) binds only to E, not to ES
Noncompetitive inhibition - inhibitor (I) binds either to E and/or to ES
Uncompetitive inhibition - inhibitor (I) binds only to ES, not to E. This is a hypothetical case that
has never been documented for a real enzyme, but which makes a useful contrast to
competitive inhibition.
Mixed inhibition-when the dissociation constants of (I) to E and ES are different. The inhibition
is mixed.
Sample Questions
What is the v/Vmax ratio when [S]=5Km
Draw a Lineweaver-Burk plot if Vmax
m=2mM.
Draw the new lineweaver-Burk plot on the same plot as above if a competitive inhibitor is
added. [I]=0.5 mM, KI=1mM.
Ribozymes
It was assumed that all enzymes are proteins until 1982 when Thomas Cech and Sydney
Altman discovered catalytic RNAs (Nobel, 1989 in Chemistry);
Catalytic RNA, or, ribozymes, satisfy several enzymatic criteria: substrate specificity, enhance
reaction rate, and emerge from reaction unchanged;
Several known ribozymes:
RNase P: catalyzes cleavage of precursor tRNA molecules into mature tRNAs;
Group I, II introns: catalyze their own splicing (cleaving and ligating);
Ribosome: catalyzes peptidyl transfer reaction
Catalytic Antibodies: Abzymes
Antibodies are immunoglobulins. Antibodies are elicited in an organizm in response to
immunological challenge by a foreign molecule called antigens;
Antibodies elicited in response to transition state analogs have the ability to stabilize the
transition state and thus can catalyze a reaction by forcing the substrate into the transition state
structure;
Examples of abzymes: How do enzymes stabilize the transition state of a reaction
General Acid-base catalysis
Metal catalysis
Covalent catalysis
Substrate strain
Catalysis by approximation.
General Acid-Base Catalysis
A molecule other than water plays the role of a proton donor or acceptor.
Ionizable groups on the protein provide the H+ transferred in the transition state. Thus an
ionizable group will be most effective as an H + transferring agent at or near its pKa. Histidine
pKa is around 7. It is the most effective general acid or base.
Example: Consider the hydrolysis of p-nitrophenylacetate with imidazole acting as a general
base.
The water has been made more nucleophilic without generation of a high concentraion of OH or without the formation of unstable, high-energy species.
Covalent catalysis
A “charge relay” increases the reactivity of Ser 195 in chymotrypsin
The specificity pocket in chymotrypsin is lined by hydrophobic residues
The different substrate specificities of the serine proteases are due to differences in their
specificity pockets
Proximity
Enzyme can hold two reactants together in an orientation that is suitable for
catalysis during a reaction in order to increase the rate of reaction.
Example: The last step of glycolysis catalyzed by pyruvate kinase
transfers a phosphate group from phophoenolpyruvate (PEP) to ADP to make ATP.
PEP and ADP are brought together by pyruvate kinase.
Enzyme activity is regulated by four different mechanisms*
(1) Allosteric control
(2) Covalent modification
(3) Proteolytic activation
(4) Stimulation or inhibition by control proteins
changes in enzyme levels due to regulation of protein synthesis or degradation are additional,
long-term ways to regulate enzyme activity
1. Allosteric = “other site” other than active site
2. Regulatory molecules called, effectors, modulators,
regulatory molecules
3. Homotropic regulation: regulation by substrate at
active site4. Heterotropic regulation: regulation by molecule
NOT substrate ( end products), at allosteric site
5. Few enzymes are allosteric
6. Allosteric enzymes DO NOT exhibit M-M kineticsAllosteric regulators do not bind to the
active site of the enzyme
Activation or inhibition of an enzyme’s activity due to binding of an activator or inhibitor at a
site that is distinct from the active site of the enzyme.
Enzymes involved in protein digestion, blood clotting, and tissue and bone remodeling are
synthesized in an inactive conformation, then activated by proteolytic cleavage
What is an isozyme?
(1) Isozymes are physically distinct forms of the same enzyme.
(2) Isozymes may differ from each other by differences in their amino acid sequences or by
the presence of different posttranslational modifications in each isozyme.
(3) The relative abundance of different isozymes varies for different tissues. The ability to
control which isozymes are expressed in a particular cell allows each cell to adjust the enzyme
activity based on the specific conditions that exist in the cell.
Lactate Dehydrogenase is composed of four monomers
Each monomer can be either heart or muscle type
Five different isozymes of lactate dehydrogenase exist: H4, H3M, H2M2, HM3, and M4
教学过程设计（要求阐明对教学基本内容的展开及教学方法与手段的应用、讨论、作业布置）：
要求学生课前掌握通读整个章节，解答老师提出的具体问题，课上以总结的形式小结。