EnzymesPresented By

Dr. Salwa Abo El-khair

3

Catalytic Proteins 5: Enzymes

Enzyme Regulation

Regulation of enzyme activity is important to

coordinate the different metabolic processes.

It is also important for homeostasis i.e. to

maintain the internal environment of the

organism constant.

Regulation of enzyme activity:

It can be achieved by two general mechanisms:

A) Control of enzyme quantity

Altering the rate of enzyme synthesis and

degradation.

Induction.

Repression.

Concentration of substrates, coenzymes and metal

ion activators.

B) Altering the catalytic efficiency of the

enzyme:

Allosteric regulation.

Feedback inhibition.

Proenzyme (zymogen).

Covalent modification.

Protein-protein interaction.

A) Control of enzyme quantity

1- Control of the rates of enzyme synthesis and

degradation.

Enzymes are protein in nature, they are

synthesized from amino acids under gene control

and degraded again to amino acids.

Enzyme quantity depends on the rate of enzyme

synthesis and the rate of its degradation.

Increased enzyme quantity may be due to an

increase in the rate of synthesis, a decrease in

the rate of degradation or both.

Decreased enzyme quantity may be due to a

decrease in the rate of synthesis, an increase in

the rate of degradation or both.

For example, the quantity of Liver Arginase

enzyme increases after protein rich meal due

to an increase in the rate of its synthesis; also

it increases in starved animals due to a

decrease in the rate of its degradation.

2- Induction

Induction means an increase in the rate of enzyme

synthesis by substances called inducers.

According to the response to inducers,

enzymes are classified into:

Constitutive enzymes, the concentration of

these enzymes does not depend on inducers.

Inducible enzymes, the concentration of these

enzymes depends on the presence of inducers.

For example, induction of lactase enzyme in

bacteria grown on glucose media.

3- Repression

Repression means a decrease in the rate of

enzyme synthesis by substances called

repressors.

Repressors are low molecular weight

substances that decrease the rate of enzyme

synthesis at the level of gene expression.

Repressors are usually end products of

biosynthetic reaction, so repression is sometimes

called feedback regulation.

For example, dietary cholesterol decreases the

rate of synthesis of HMG CoA reductase (β-

hydroxy β-methyl glutaryl CoA reductase), which

is a key enzyme in cholesterol biosynthesis.

4- Derepression

Following removal of the repressor or its

exhaustion, enzyme synthesis retains its normal

rate.

5- Concentration of substrates, coenzymes and

metal ion activator:

The susceptibility of enzyme to degradation

depends on its conformation.

Presence of substrate, coenzyme or metal

ion activator causes changes in the enzyme

conformation decreasing its rate of

degradation.

AP Biology 2007-2008

Got any Questions?!

Activity Enumerate ways to control quantity of

enzyme and discuss one of them

Discuss feed back regulation (def, mechanism

,example)

B) Control of catalytic efficiency of enzymes

1- Allosteric Regulation

Allosteric enzyme is formed of more than

one protein subunit.

It has two sites; a catalytic site for substrate

binding and another site (allosteric site), that is

the regulatory site, to which an effector binds.

Allosteric regulation

Allosteric means “other site”

E

Active site

Allosteric

site

Allosteric means another site

If binding of the effector to the enzyme

increases it activity, it is called positive effector

or allosteric activator e.g. ADP is allosteric

activator for phosphofructokinase enzyme.

If binding of the effector to the enzyme causes

a decrease in its activity, it is called negative

effector or allosteric inhibitor e.g.

ATP and citrate are allosteric inhibitors for

phosphofructokinase enzyme.

Glucose-6-phosphate is allosteric inhibitor for

hexokinase enzyme.

The allosteric site the enzyme “on-

off” switch

E

Active

site

Allosteric

site emptySubstrate

fits into

the active

site

The inhibitor

molecule is

absent

Conformational

change

Inhibitor fits

into allosteric

site

Substrate

cannot fit

into the

active site

Inhibitor

molecule

is present

E

Mechanism of allosteric regulation

Binding of the allosteric effector to the

regulatory site causes conformational changes

in the catalytic site, which becomes more fit for

substrate binding in positive effector (allosteric

activator), and becomes unfit for substrate

binding in negative effector (allosteric inhibitor)

AP Biology

Allosteric regulation

Conformational changes by regulatory molecules

inhibitors keeps enzyme in inactive form

activators keeps enzyme in active form

Conformational changes Allosteric regulation

Conformational changes Allosteric regulation

2- Feedback Inhibition

In biosynthetic pathways, an end product may

directly inhibit an enzyme early in the pathway.

This enzyme catalyzes the early functionally

irreversible step specific to a particular biosynthetic

pathway.

Feedback inhibition may occur by simple

feedback loop.

AP Biology End product is an inhibitor of enzyme 1

Feedback Inhibition

final product is inhibitor of earlier step

A B C D E F Genzyme

1

enzyme

2

enzyme

3

enzyme

4

enzyme

5

enzyme

6

X

Feedback inhibition can occur by multiple

feedback inhibition loops as occurs in branched

biosynthetic pathways.

Feedback regulation is different from

feedback inhibition.

Feedback regulation:

It means that an end product in the reaction

decreases the rate of enzyme synthesis at the level

of gene expression.

It decreases the enzyme quantity through the

action on the gene that encodes the enzyme.

It does not affect the enzyme activity.

It is a complicated process that takes hours to

days.

For example, inhibition of HMG CoA reducatse

enzyme by dietary cholesterol.

Feedback inhibition

It means that an end product directly inhibits

an enzyme early in biosynthetic pathways.

It does not affect enzyme quantity.

It decreases the enzyme activity.

It is a direct and rapid process that occurs in

seconds to minutes.

For example, CTP inhibits aspartate

transcarbamylase enzyme in pyrimidine synthesis.

AP Biology 2007-2008

Got any Questions?!

Activity 1- During ___________the final product of a metabolic

pathway turn off the first step of metabolic pathway.

(A) Positive feed back

(B) Negative feed back

(C) Competitive feed back

(D) Both A and C

2- An allosteric modulator influences enzyme activity by

(A) Competing for the catalytic site with the substrate

(B) Binding to a site on the enzyme molecule far from the

catalytic site

(C) Changing the nature of the product formed

(D) Covalently modifying enzyme

Activity Enumerate factors control catalytic activity of

enzyme and discuss one of them

Discuss allosteric regulation (def, mechanism

,types )

Compare between feedback regulation and

feed back inhibition

3- Proenzymes (Zymogens)

Some enzymes are secreted in inactive forms

called proenzymes or zymogens.

Examples for zymogens include:

1. Pepsinogen,

2. Trysinogen,

3. Chymotrypsinogen,

4. Prothrombin and Clotting factors.

Zymogen is inactive because it contains an

additional polypeptide chain that masks (blocks)

the active site of the enzyme.

Activation of zymogen occurs by removal of

the polypeptide chain that masks the active site.

Activation of chymotrypsinogen to chymotrypsin, and of trypsinogen to trypsin

Activation of zymogens can occur by one of

the following methods:

a) Activation by HCl

HCl

Pepsinogen Pepsin

b) Activation by other enzymes

Enterokinase

Trypsinogen Trypsin

Thrombokinase + Ca++

Prothrombin Thrombin

c) Auto activation i.e. the enzyme activates

itself.

Pepsin

Pepsinogen Pepsin

Biological importance of zymogens

Some enzymes are secreted in zymogen from to

protect the tissues of origin from auto digestion.

To insure rapid mobilization of enzyme activity

at the time of needs in response to physiological

demands.

4- Protein-protein interaction

In enzymes that are formed from of many protein

subunits, the enzyme may be present in an inactive

from through interaction between its protein

subunits.

The whole enzyme, formed of regulatory and

catalytic subunits, is inactive.

Activation of the enzyme occurs by separation of

the catalytic subunits from the regulatory subunits.

• Protein kinase A enzyme is an example for

regulation of enzyme activity by protein interaction.

• It is formed of 4 subunits, 2 regulatory (2R) and 2

catalytic (2C) subunits.

• The whole enzyme (2R2C) is inactive.

• cAMP (cyclic adenosine monophosphate) activates

the enzyme by binding to the 2 regulatory (2R)

subunits releasing the 2 catalytic (2C) subunits.

5- Covalent modification

It means modification of enzyme activity through

formation of covalent bonds e.g.

Methylation (addition of methyl group).

Hydroxylation (addition of hydroxyl group).

Adenylation (addition of adenylic acid).

Phosphorylation (addition of phosphate group).

Reversible covalent modification

What’s covalently modulated enzymes?

•Activity is modulated by covalent

modification of one or more of its amino

acid residues in the enzyme molecule.

• Common modifying groups include:

phosphoryl, adenylyl, methyl and

hydroxyl.

• These groups are generally linked to

and removed from the regulatory

enzyme by separate enzymes.

Phosphorylation is the most covalent

modification used to regulate enzyme activity.

Phosphorylation of enzyme occurs by addition of

phosphate group to the enzyme at the hydroxyl

group of serine, threonine or tyrosine.

This occurs by protein kinase enzyme.

Protein kinases catalyze the

phosphorylation of proteins

Dephosphorylation of the enzyme occurs by

removal of phosphate group from the hydroxyl

group of serine, threonine or tyrosine.

This occurs by phosphatase enzyme.

Protein phosphatases remove phosphate

groups from phosphorylated proteins

OH

++

O P O-O-

O

ATP ADP

Pi

ProteinKinase

Protein Phosphatase

•Phosphorylation and dephosphorylation are not the reverse of one another.

• The rate of cyclingbetween the phosphorylated and the dephosphorylated states depends on the relative activities of kinases and phosphatases.

The phosphorylated from is the active form in

some enzymes, while the dephosphorylated form

is the active form in other enzymes.

(active form)

(active form)

(inactive form)

(inactive form)

Enzymes activated by phosphorylation:

These are usually enzymes of degradative

(breakdown) reactions e.g.

1. Glycogen phosphorylase that breaks down

glycogen into glucose.

2. Citrate lyase, which breaks down citrate.

3. Lipase that hydrolyzes triglyceride into glycerol

and 3 fatty acids.

Enzymes inactivated by phosphorylation:

These are enzymes of biosynthetic reactions

1. Glycogen Synthetase, which catalyzes

biosynthesis of glycogen.

2. Acetyl CoA carboxylase, an enzyme in fatty

acid biosynthesis.

3. HMG CoA reductase, an enzyme in cholesterol

biosynthesis.

AP Biology 2007-2008

Got any Questions?!

Activity 1- The inactive precursor of an active enzyme is called

A) Zymogen

B) Ribozyme

C) Isozyme

D) Apoenzyme

2- Activation or inactivation of certain key regulatory

enzymes is accomplished by covalent modification of

the amino acid:

(A) Alanine

(B) Lysine

(C) Phenylalanine

(D) Serine

Activity Discuss protein- protein interaction ( mechanism

,one example )

Discuss proenzyme and its biomedical importance

Enumerate methods of covalent modification of

enzyme activity

Discuss covalent modification by phosphorylation

(types, enzymes involved, examples)

Isoenzymes

Isoenzymes (isozymes) are multiple forms of the

enzyme that have the same catalytic activity.

Although they have the same catalytic activity,

they are physically distinct and differ in

electrophoretic mobility and liability to inhibitors.

Iso means the same and isoenzyme means the

same enzyme.

Example of isoenzymes

Many enzymes are present in isoenzyme form:

1. Lactate dehydrogenase

2. Creatine kinase

3. Acid phosphatase

4. Alkaline phosphatase

Lactate dehydrogenase (LDH)

It is an enzyme that catalyzes the removal of 2

hydrogen atoms from lactic acid forming pyruvic

acid.

Lactate dehydrogenase

Lactic acid Pyruvic acid

NAD NADH+H

Its level in plasma increases in:

1. Myocardial infarction (heart diseases).

2. Viral hepatitis (liver disease).

3. Leukaemia (blood disease).

LDH enzyme is a tetramer formed of 4 protein

subunits; each subunit is called protomer.

The protomers of LDH are of 2 types, H (after

heart) and M (after muscle).

LDH isoenzymes are clinically important to

differentiate between heart, liver and blood

diseases.

LDH has 5 isoenzymes:

LDH1 is formed of HHHH. It increases in myocardial infarction.

LDH2 is formed of HHHM. It increases in myocardial infarction.

LDH3 is formed of HHMM. It increases in leukaemia.

LDH4 is formed of HMMM. It increases in viral hepatitis.

LDH5 is formed of MMMM. It increases in viral hepatitis.

HHHH

MHHH

MMHH

MMMM

MH Heart type Muscle type

MMMH

Creatine kinase (CK)

It is an enzyme that catalyzes phosphorylation of

creatine.

Creatine Kinase

Creatine Creatine phosphate

ATP ADP

Its level in plasma increases in

1. Brain tumors.

2. Myocardial infarction (heart disease).

3. Skeletal muscle diseases.

CK isoenzymes are clinically important to

differentiate between brain, heart and

skeletal muscle diseases.

CK enzyme is a dimmer formed of 2 protein

subunits (protomers), B (after brain) and M (after

muscle).

CK has 3 isoenzymes:

CK BB which increases in brain tumors.

CK MB which increases in heart diseases.

CK MM which increases in skeletal muscle diseases.

Source of isoenzymes

Isoenzymes may be produced by the same gene

but the subunits undergo different post-

translation modifications in different organs.

Isoenzymes may be produced by more than one

gene; each gene produces one subunit.

Medical importance of isoenzymes

Isoenzymes are not only important for diagnosis

but also indicate the diseased organ.

Lactate dehydrogenase enzyme (LDH) increases

in myocardial infarction (heart disease), viral

hepatitis (liver disease) and leukaemia (blood

disease).

LDH isoenzymes indicate the diseased organ:

LDH1 and LDH2 isoenzymes increase only in

myocardial infarction,

LDH3 increases in leukaemia

LDH4 and LDH5 increase in viral hepatitis.

Antienzymes

These are substances secreted by living cells or

organisms that inhibit enzyme activity e.g.:

Ascaris worms living in the intestine secrete

antienzymes (anti-trypsin and anti-pepsin) so;

they are not digested by proteolytic enzymes

present in the digestive juices.

Mucin lining the stomach contains antienzyme

(anti-pepsin) that prevents digestion of stomach

wall by pepsin.

Blood plasma contains natural antienzyme

(anti-thrombin) that inactivates thrombin after

blood coagulation to prevent its intra-vascular

spreading.

Ribozymes

Ribozymes are enzymes but they are not protein in

nature, they are nucleic acid in nature formed of

RNA.

Ribozymes catalyze cleavage of RNA by

hydrolysis of phosphate diester bonds e.g.

cleavage of pre-mRNA to form mRNA.

AP Biology 2007-2008

Got any Questions?!

Activity 1- The enzyme Creatine kinase levels are increased in the

blood of patients with

A) Prostate cancer .

B) Hepatitis

C) Heart attack

D) Osteoporosis

2- The isoenzymes of LDH

(A) Differ only in a single amino acid

(B) Differ in catalytic activity

(C) Exist in 5 forms depending on M and H monomer

contents

(D) Occur as monomers

Activity Discuss antienzymes (def , examples , biomedical

importance).

Discuss iso-enzymes (def, 2 examples & clinical

importance).

Creatine kinase & Lactate dehydrogenase can be

used for diagnosis and follow up of some diseases

(explain )

Thank you