Download 5-MGD Session 3, Lec 5, 2014

Molecule, Gene, and disease
Sun. 2 – 3 – 2014
Session 3
Enzymes and enzyme regulation
Dr. Muna A. R.
Structure of Session 3
Lecture 5:
Enzyme activity: kinetics and inhibition
8:00 – 8:50
Lecture 6:
Regulation of enzyme activity
8:50 – 9:40
Work Session 2
10:00 – 12:00
Learning outcomes
1) Explain the effects of enzymes on chemical reactions.
2) Describe how reaction rates vary as a function of enzyme and
substrate concentration.
3) Define the terms activity, international unit of enzyme activity, Km
and Vmax.
4) Analyse and interpret kinetic data for enzyme-catalysed reactions.
5) Describe the effects of enzyme inhibitors on enzyme kinetics and be
able to distinguish between the two from simple graphs.
6) List the major regulatory mechanisms that control enzyme activity
(plus examples).
Learning outcomes
7) Discuss the allosteric properties of a key regulatory enzyme such as
phosphofructokinase.
8) Discuss the concept of enzyme cascades and the use of protein
kinases and phosphatases to regulate activity.
9) Define the term zymogen and give examples of enzymes that are
derived from zymogens.
10) Explain how activation of the clotting cascade leads to the
formation of a fibrin clot.
11) Discuss the mechanisms that are involved in the regulation of clot
formation and breakdown.
Suggested reading
• Marks’ Basic Medical Biochemistry Chapters 8,
9, 45
• Medical Biochemistry Chapters 6, 7
• Lippincott’s Illustrated Reviews: Biochemistry
Chapter 5
Nomenclature
Enzymes are named by the type of reaction that
they catalyse. Usually this means adding the
suffix –ase to the name of their
substrate or reaction that they catalyse
e.g. Lactase hydrolyses lactose into glucose and
galactose
DNA polymerase, polymerises deoxynucleotides
to form DNA
Six Major Classes of Enzymes
Six Major Classes of Enzymes
Enzyme Commission : established the nomenclature
for enzymes
• numbers that follow E.C. gives the identity of the
enzyme
Properties of enzymes
1. Virtually all enzymes are proteins
Some enzymes also require the presence of additional
chemical components to catalyse reactions.
*Cofactors are inorganic ions such as Fe2+, Mn2+etc.
*Coenzymes are organic compounds that act as temporary
carriers of groups in the reaction e.g. nicotinamide
adenine dinucletide (NAD), Coenzyme A (CoA).
*Coenzymes or cofactors that are tightly or covalently
linked to the enzyme protein are known as prosthetic
groups.
2. Enzymes are highly specific
Interact with one or only a few substrates and catalyse one
type of reaction.
• The protein part of the enzyme
(apo-enzyme) +Cofactor,prosthetic group,or the
coenzyme= Holoenzyme
Properties of enzymes
3. Enzymes increase the rate of a reaction
Enzymes increase the rate of the reaction by factors of
1 million or more. They DO NOT affect the
equilibrium of a reaction.
4. Enzymes are left unchanged after the reaction has
occurred.
Enzymes provide a place for the reaction occur.
Although there may be changes to the enzyme during
the course of the reaction on completion the enzyme
will be unchanged.
How do enzymes work?
Enzymes work by lowering the
activation energy needed for a
reaction to occur. Binding of
substrate to a distinct part of the
enzyme,
increases
the
active
the
site,
local
concentration of reactants and
also stabilises the formation of
the high energy transition state.
The active site
The active site of an enzyme is the place where the reaction occurs.
Only
molecules that have a complementary shape to the active site will be able to bind
(Lock and Key Hypothesis). The substrate is held in the active site by multiple
weak bonds with amino acids in this part of the enzyme.
The Michaelis-Menten Model :
E+S
ES
E + P
Vo = Vmax [S]
[S] + Km
WhereV0= initial reaction velocity
[S]
= substrate concentration
Vmax = maximal velocity
Km
= Michaelis constant
*Low Km means high affinity of the
enzyme to the substrate.
*High Km means low affinity of the
enzyme to the substrate.
Enzymes – Lineweaver-Burk Equation
V = Vmax [S]
[S] + Km
Inverting the Equation yields:
1 = Km
+ 1
V Vmax [S]
Vmax
Inverting the Equation yields:
(Lineweaver-Burke Equation)
By plotting 1/ V as a function of 1/[S],
a linear plot is obtained:
Slope = Km/Vmax
y-intercept = 1/Vmax
FACTORS AFFECTING REACTIO N VELOCIT Y
1) Substrate concentration
FACTORS AFFECTING REACTIO N VELOCITY
FACTORS AFFECTING REACTIO N VELOCIT Y
2) Effect of Temp.
3) Effect of pH
Inhibition of enzyme activity
Many drugs work by inhibiting the activity of enzymes.
1-Irreversible inhibitors:
Bind covalently to the enzyme molecule to destroy
enzyme function
2-Reversible inhibitors
i) Competitive inhibitors: -Binds at the active site
-Affects Km not Vmax
-Can be overcome by increasing the substrate concentration
Examples of competitive inhibitors:
*Allopurinol
competes
with
hypoxanthine
for
xanthine oxidase inhibiting the formation of uric acid,
so it is used in treatment of hyperuricemia (gout).
*Statins (e.g. atorvastatin) competes with HMGCoA
for its reductase, so, it inhibits cholesterol synthesis.
ii) Non-competitive inhibitors:
-Binds at a site other than
the active site
-Affects Vmax not Km
-Cannot be overcome by
increasing the substrate
concentration.
Non-competitive inhibitors