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 CommissionEnzyme Commission : established the nomenclature for enzymes

• numbers that follow E.C. gives the identity of the enzyme

Properties of enzymes1. 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

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.

 

 

Properties of enzymes

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, the active site,

increases the 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] + KmWhereV0= 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] + KmInverting 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/Vmaxy-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