FIRST BIOCHEMISTRY EXAM - PowerPoint Presentation

Slide1

FIRST BIOCHEMISTRY EXAM

Tuesday 25/10/2016

10-11

40 MCQs.

Location : 102, 105, 106, 301, 302

Slide2

The Behavior of Proteins: Enzymes,

Mechanisms, and Control

Slide3

General theory of enzyme action, by

Leonor

Michaelis

and Maud

Menten in 1913.

Leonor

Michaelis

, German1875–1949

Maud

Menten

, Canadian

1879–1960

Slide4

Learning Objectives

1.

What Is the

Michaelis–Menten

Approach to Enzyme

Kinetics?

2. How Do Enzymatic Reactions Respond to Inhibitors?3. Does the Michaelis–Menten Model Describe the Behavior of

Allosteric Enzymes ? 4. What Are the Models for the Behavior of Allosteric Enzymes?

5. What is

Lineweaver

–Burk plot ?

5 . How Does

Phosphorylation

of Specific Residues Regulate

Enzyme Activity?

6. What Are Zymogens, and How Do They Control Enzyme Activity?

7. What Are Coenzymes and Cofactors ?

8. What Are

Isoenzymes

& what is their clinical application.

Slide5

They postulated that the enzyme first combines reversibly with its substrate to form an enzyme-substrate complex in a relatively fast reversible step:

The ES complex then breaks down in a slower second step to yield the free enzyme and the reaction product P:

Slide6

Slide7

Michaelis-Menten Model

2

V

max

[S]

V

=

K

M

+ [S]

Michaelis-Menten

equation

Slide8

Michaelis-Menten Model

When [S]= K

M

, the equation reduces to

2

Slide9

Michaelis-Menten Model

It

is difficult to determine

V

max experimentally

The equation for a hyperbola (E saturation curve)

Can be transformed into the equation for a straight line by taking the reciprocal of each side

Slide10

The physiological consequence of

K

M

is illustrated by the sensitivity of some individuals to

ethanol

. Such persons exhibit facial flushing and rapid heart rate (tachycardia) after ingesting even small amounts of alcohol.In the liver, alcohol

dehydrogenase converts ethanol into acetaldehyde

Slide11

Normally, the acetaldehyde, which is the cause of the symptoms when present at high concentrations, is processed to acetate by acetaldehyde

dehydrogenase

.

Slide12

Most people have two forms of the acetaldehyde

dehydrogenase

,

a low

K

M mitochondrial form and a high KM

cytosolic

form. In susceptible persons, the mitochondrial enzyme is less active due to the substitution of a single amino acid, and acetaldehyde is processed only by the cytosolic enzyme. Because this enzyme has a high K

M

, less acetaldehyde is

converted into acetate; excess

acetaldehyde

escapes into the blood and accounts for the physiological effects.

Slide13

Lineweaver

-Burk Plot

which has the form y =

mx

+ b, and is the formula for a straight line (

linearization)

a plot of 1/V versus 1/[S] will give a straight line with slope of KM/

Vmax and y intercept of 1/Vmaxsuch a plot is known as a Lineweaver

-Burk double reciprocal

plot

Slide14

Lineweaver

-Burk Plot

K

M

is the dissociation constant for ES; the

greater

the value of KM

, the less tightly S is bound (less affinity) to E

The

units K

M

are units of [S] that is M or

mM

Slide15

Turnover number

Turnover number (

k

cat

): is the moles of

S converted to P per mole of

E per second.Unit of k

cat is mol S*mol E-1*s-1

The higher

K

cat

value the higher the reactivity of the E

Slide16

Turnover Numbers and K

M

Values for some typical enzymes

Enzyme

Function

Catalase

C

onversion of

H

2

O

2

to H

2

O + O

2

4 x 10

7

25

Carbonic

anhydrase

Hydration of CO

2

1 x 10

6

12

Acetylcholin-

esterase

Regeneration

of acetylcholine

1.4 x 10

4

9.5 x 10

-2

Chymotrypsin

Proteolytic enzyme

1.9.x 10

2

6.6 x 10

-1

Lysozyme

Hydrolysis of

bacterial cell wall

polysaccharides

0.5

6 x 10

-3

K

M

(mmol•liter

-1

)

Turnover numbr

[(mol S)•(mol E)

-1

•s

-1

]

Slide17

Enzyme Inhibition

Reversible inhibitor

:

a substance that binds to an enzyme to inhibit it, but can be released

competitive inhibitor

:

binds to the active (catalytic) site and blocks access to it by substrate

noncompetitive inhibitor:

binds to a site other than the active site; inhibits the enzyme by changing its conformation

Irreversible inhibitor

:

a substance that causes inhibition that cannot be reversed

usually involves formation or breaking of covalent bonds to or on the enzyme

Cyanides, Penicillin, Heavy metals, Nerve gas

Slide18

competitive inhibitor

A competitive inhibitor diminishes the rate of

catalysis by reducing the proportion of enzyme molecules bound to a substrate. At any given inhibitor concentration,

competitive inhibition can be relieved by increasing the substrate concentration. Under these conditions, the substrate "outcompetes" the inhibitor for the active site.

Slide19

Methotrexate

is a structural analog of

tetrahydrofolate

, a coenzyme for the enzyme

dihydrofolate reductase, which plays a role in the biosynthesis of purines

and pyrimidines . It binds to dihydrofolate

reductase 1000-fold more tightly than the natural substrate and inhibits nucleotide base synthesis. It is used to treat cancer.

Slide20

The cofactor

tetrahydrofolate

and its structural analog

methotrexate

. Regions with

structural differences are shown in red.

Slide21

noncompetitive inhibitor

In

noncompetitive inhibition, which also is reversible, the inhibitor and substrate can bind simultaneously to an enzyme

molecule at different binding sites . A noncompetitive inhibitor acts by decreasing the turnover number rather than by diminishing the proportion of enzyme molecules that are bound to substrate. Noncompetitive inhibition, in

contrast with competitive inhibition, cannot be overcome by increasing the substrate concentration.

Slide22

Competitive

Vs

noncompetitive Inhibition

Slide23

Competitive Inhibition Illustrated on a Double-Reciprocal Plot. A double-reciprocal plot of enzyme kinetics in the presence

( I )

and absence ( I ) of a competitive inhibitor illustrates that the inhibitor has no effect on

V max

but increases

K M.

Slide24

Noncompetitive Inhibition

Illustrated on a Double-Reciprocal Plot. A double-reciprocal plot of enzyme kinetics in the presence

( I )

and absence ( I ) of a noncompetitive inhibitor shows that

K M

is unaltered and

V max

is decreased.

Slide25

Irreversible inhibitor

Penicillin acts by covalently modifying the enzyme

transpeptidase

, thereby preventing the synthesis of bacterial cell walls and thus killing the bacteria .

Aspirin acts by covalently modifying the

enzyme

cyclooxygenase, reducing the synthesis of inflammatory signals.

Slide26

Structure of Penicillin

Formation of a

Penicilloyl

-Enzyme Complex.

Slide27

Chymotrypsin

Inhibition

Treatment with

organofluorophosphates

such as

diisopropylphosphofluoridate

(DIPF) was found to inactivate the enzyme irreversibly .Despite the fact that the enzyme possesses 28 serine residues, only one, serine 195

, was modified, resulting in a total loss of enzyme activity. This chemical modification reaction suggested that this unusually reactive serine residue plays a central role in the catalytic mechanism of chymotrypsin.

Slide28

An Unusually Reactive Serine in

Chymotrypsin

.

Chymotrypsin

is inactivated by treatment with

diisopropylphosphofluoridate

(DIPF), which reacts only with serine 195 among 28 possible serine residues

Slide29

Enzyme regulation

Enzyme activity can be regulated by several ways:

Covalent modification (

Phosphorylation

)

The

side chain -OH groups of Ser,

Thr

, and Tyr can form phosphate esters

phosphorylation

by ATP

can convert an inactive precursor into an active enzyme

Protein

kinases

phosphorylate

enzymes

Protein

phosphatases

remove phosphate groups

Slide30

Enzyme regulation

Covalent modification (

Phosphorylation

)

Slide31

Noncovalent

modification (

Allosteric

regulation)

Using

allosteric

effectors (activator and

allosteric inhibitor )

Control amount of enzyme:

enzyme synthesis: gene regulation

enzyme degradation

Slide32

Zymogen

: an inactive precursor of an enzyme; cleavage of one or more covalent bonds transforms it into the active enzyme

Involved in blood clotting and digestion

Example

chymotrypsinogen

Chymotrypsinogen

synthesized and stored in the pancreasa single polypeptide chain of 245 amino acid residues cross linked by five disulfide (-S-S-) bondsenzyme trypsin

cleaves

it to give

chymotrypsin

Slide33

Secretion of Zymogens by an

Acinar

Cell of the Pancreas

Slide34

Proteolytic

Activation of

Chymotrypsinogen

. The three chains of

α

-chymotrypsin are linked by two interchain disulfide bonds

Slide35

Zymogen

Activation by

Proteolytic

Cleavage

Active enzymes are shown in yellow

Slide36

Blood-Clotting Cascade

Slide37

Many Enzymes Require Cofactors for Activity

Slide38

COENZYMES

……

Cofactors

Slide39

Enzyme cofactors

Metal

Enzyme

Zn2+

Carbonic

anhydrase

Zn2+

Carboxypeptidase

Mg2+

Hexokinase

Ni2+

Urease

Se

Glutathione

peroxidase

Mn2+

Superoxide dismutase

Cu2

Cytochrome

oxidase

Slide40

ISOENZYMES

Isozymes

or

isoenzymes

, are enzymes that differ in amino acid sequence yet catalyze the same reaction. Usually, these

enzymes display different kinetic parameters, such as K M, or different regulatory properties.

They are encoded by different genetic loci, which usually arise through gene duplication and divergence.

Slide41

ISOENZYMES

The existence of

isozymes

permits the fine-tuning of metabolism to meet the particular needs of a given tissue or developmental stage. Consider the example of lactate

dehydrogenase

(

LDH), an enzyme that functions in anaerobic glucose metabolism and glucose synthesis. Human beings have two isozymic

polypeptide chains for this enzyme: the H isozyme highly expressed in heart and the M

isozyme

found in

skeletal muscle

. The amino acid sequences are 75% identical.

Slide42

The functional enzyme is

tetrameric

, and many different combinations of the two subunits are possible. The

H4

isozyme, found in the heart, has a higher affinity for substrates than does the M4 isozyme

. The two isozymes also differin that high levels of pyruvate

allosterically inhibit the H4 but not the M4 isozyme. The other combinations, such as H3M, have intermediate properties depending on the ratio of the two kinds of chains.

Slide43

ISOENZYMES……… LDH

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END

Chapter 7