principles of biochemistry (semester 2 -2014/15) · principles of biochemistry (semester 2...

BCH 3000PRINCIPLES OF BIOCHEMISTRY

(Semester 2 -2014/15)

1

Enzymology -1

2

Enzymology -2

11

Catalysis and Enzymology

• Introduction to catalysis

– Free energy profiles

– What enzymes do

• Chemical kinetics and reaction order

– First-order kinetics

– Other reaction orders

• Enzyme kinetics

– Binding and saturation

12

What is a catalyst?

• Example:

Features of a catalyst:

• Makes an alternative reaction path in which less

activation energy (DG‡) is needed

• Equally increases the rate of the back and forth

reaction reaction catalysis has no effect on

equilibrium position!

H3C C

O

OH + C2H5OH H3C C

O

OC2H5 + H2OH+

“A catalyst is a compound which enhances the rate of a

chemical reaction without being destroyed or incorporated in

the product” (IUPAC)

13

DG‡

Gibbs

free

energy

S

P

Reaction coordinate

X‡ (T.S.)

14

Two ways to decrease DG‡:

a By lowering the energy content

of the transition state

Eact

Eact

E

progress of reaction

a b

b By ground state

destabilisation15

What kinds of catalysts are there

around?• Organic

• Inorganic

• Biological (biocatalyst)

Other way of subdivision:

• Homogeneous = freely dissolved in solution

– organic catalyst

– organometallic complex

– enzyme in water

• Heterogeneous = solid, in liquid or gaseous

environment

– inorganic catalyst (e.g. zeolite)

– immobilised enzyme

– enzyme in an organic solvent16

All Enzymes are not Proteins:

Ribozymes

• Ribozymes defined as RNA catalysts

• Function as phosphodiester

transferases

– Sequence specific

– Kinetically like enzymes

– Efficiently lower DG*

– Require native 3D-structure

• Function in RNA splicing

17

How Enzymes Work

• Enzymes are protein catalysts: they

increase the velocity of the reaction, but

are not themselves altered

• Enzymes cannot change Keq/DGo

• Enzymes do change DG*/k

– Energy of transition state decreased or

– Energy of ground state increased

18

Simple Kinetics: A First-order

Reaction

A B

v = -d[A]/dt = d[B]/dt units: M/time

v = k[A] Empirical rate law

(M x time-1) = k(M) units of k: time-1

Reaction order: exponent of concentration

term on which rate depends. Above rxn is 1st

order in reactant A 19

1st-Order Rate Equation

v = -d[A]/dt = k[A]

or d[A]/[A] = d ln[A] = -kdt

so, ln [A] = ln[Ao] –kt and [A] = [Ao]e-kt

Slope = -k[A]=[Ao]e-kt;

exponential

decay

20

Other Reaction Orders

• Zero order reaction independent of [A]

– v = k, units of k: M x time -1

• Second order reaction: A + B P

– v = k[A][B]

– k: unit of M-1time-1

– 1st order in A

– 1st order in B; second order overall

– For 2A P; v = k[A]2; 2nd order in A

21

Order in Enzyme Catalysis

depends on [S]

S P

vo = initial velocity; measure at

several [S] but constant [E]

25

Michaelis-Menten Kinetics. I.

At infinite [S], vo = Vmax

Km defined as [S] at which vo = ½ Vmax

M-M Equation:

vo = (Vmax [S])/(Km + [S])

26

Michaelis-Menten Kinetics. II

if [S] << Kmvo = (Vmax/Km)[S]

if [S] = Km vo = 0.5 Vmax

if [S] >> Km vo = Vmax

27

-10

-5

0

5

10

15

20

-50 -30 -10 10 30 50

v, µ

mo

l/m

in

[S], mM

Enzymes

ES ES P v Vmax[S]

Km [S]

k1

k-1

k2

-Km, Vmax

0

1

2

3

4

5

0 10 20 30 40 50v

, µ

mo

l/m

in

[S], mM

Km

0.5Vmax

28

Enzymology -3

32

33

Amino Acids, Proteins, and

Enzymes

Enzymes

Enzyme Action

Factors Affecting Enzyme Action

Enzyme Inhibition

33

34

Enzymes

• Catalysts for biological reactions

• Most are proteins

• Lower the activation energy

• Increase the rate of reaction

• Activity lost if denatured

• May be simple proteins

• May contain cofactors such as metal ions

or organic (vitamins)

34

35

Name of Enzymes

• End in –ase

• Identifies a reacting substance

sucrase – reacts sucrose

lipase - reacts lipid

• Describes function of enzyme

oxidase – catalyzes oxidation

hydrolase – catalyzes hydrolysis

• Common names of digestion enzymes still use –in

pepsin, trypsin 35

36

Classification of Enzymes

Class Reactions catalyzed

• Oxidoreductoases oxidation-reduction

• Transferases transfer group of atoms

• Hydrolases hydrolysis

• Lyases add/remove atoms

to/from a double bond

• Isomerases rearrange atoms

• Ligases combine molecules

using ATP 36

37

Examples of Classification of

Enzymes

• Oxidoreductoases

oxidases - oxidize ,reductases – reduce

• Transferases

transaminases – transfer amino groups

kinases – transfer phosphate groups

• Hydrolases

proteases - hydrolyze peptide bonds

lipases – hydrolyze lipid ester bonds

• Lyases

carboxylases – add CO2

hydrolases – add H2O 37

38

Enzyme Action:

Lock and Key Model

• An enzyme binds a substrate in a region called the active site

• Only certain substrates can fit the active site

• Amino acid R groups in the active site help substrate bind

• Enzyme-substrate complex forms

• Substrate reacts to form product

• Product is released

38

39

Lock and Key Model

+ +

E + S ES complex E + P

S

P

P

S

39

40

Enzyme Action:

Induced Fit Model

• Enzyme structure flexible, not rigid

• Enzyme and active site adjust shape to

bind substrate

• Increases range of substrate specificity

• Shape changes also improve catalysis

during reaction

40

41

Enzyme Action:

Induced Fit Model

E + S ES complex E + P

S

P

P

SS

41

42

Factors Affecting Enzyme

Action: Temperature

• Little activity at low temperature

• Rate increases with temperature

• Most active at optimum temperatures

(usually 37°C in humans)

• Activity lost with denaturation at high

temperatures

42

43


Action

Optimum temperature

Reaction

Rate

Low High

Temperature43

44


Action: Substrate Concentration

• Increasing substrate concentration

increases the rate of reaction (enzyme

concentration is constant)

• Maximum activity reached when all of

enzyme combines with substrate

44

45


Action

Maximum activity

Reaction

Rate

substrate concentration

45

46


Action: pH

• Maximum activity at optimum pH

• R groups of amino acids have proper

charge

• Tertiary structure of enzyme is correct

• Narrow range of activity

• Most lose activity in low or high pH

46

47


Action

Reaction

Rate

Optimum pH

3 5 7 9 11

pH

47

48

Enzyme Inhibition

Inhibitors

• cause a loss of catalytic activity

• Change the protein structure of an enzyme

• May be competitive or noncompetitive

• Some effects are irreversible

48

49

Competitive Inhibition

A competitive inhibitor

• Has a structure similar to

substrate

• Occupies active site

• Competes with substrate for

active site

• Has effect reversed by increasing

substrate concentration

49

50

Noncompetitive Inhibition

A noncompetitive inhibitor

• Does not have a structure like substrate

• Binds to the enzyme but not active site

• Changes the shape of enzyme and active

site

• Substrate cannot fit altered active site

• No reaction occurs

• Effect is not reversed by adding substrate

50

Enzymology -4

51

• The Michaelis-Menten Equation

• Louis Michaelis and Maude Menten's theory

• It assumes the formation of an enzyme-substrate complex

• It assumes that the ES complex is in rapid equilibrium with free enzyme

• Breakdown of ES to form products is assumed to be slower than 1) formation of ES and 2) breakdown of ES to re-form E and S

52

• Enzymes are regulated:

• at genetic level (transcription, translation);

• by concentration of substrate and product;

• allosterically.

74

Enzymology -5Co-enzymes &

Cofactors

75

Cofactors are exogenous molecules that

associate with proteins to yield full activity. In

the absence of cofactor, protein is an

apoprotein.

Cofactors

Prosthetic groups are covalently attached to the

protein. Examples are heme, in hemoglobin, and

riboflavin, in flavoproteins.

Co-enzymes are soluble and associate transiently

with enzyme during catalytic cycle. An example is

vitamin K in activation of blood clotting enzymes.

77

Some Cofactors

OP

OHO

HO

N

O

OH

H

N N

S OP

HO O

O POH

OH

O

NH2

N S

HN

NH

O

OH

O+

Thiamin Diphosphate Pyridoxal PhophatesBiotin

N

OH

OHN

OH

NO

O

HN

O

PO

O

O-

PO

O

O-

O

HO

N

OH

N

N

NH2N

Na+Na+

N

NN

N

OHO

O

OH

Fe

FAD

Heme

HO

ON

OH

P

O

OH

O P

O

OH

OHO

OO

N

N N

N

NH2

O

R

N

OH

OHN

OH

NO

O

HN

O

PO-

O

O-

FMN

R= H NADHR= -PO3H2 NADPH

O

NH2

N

N

N

NH2

N

O

OP

OH

O

O

OPO

OH

OH

O

HN

HN

O

SH

OHO

OH

PHO

O

Coenzyme A (CoA)

NN

N N

H2N

O NH2

O

NH2

NH2O

O

OCo

O

H

H2N

O

NH

O

C

PO

O

-OO

N

N

HO H

H

NH2

H

H

OH

N

Vitamin B12

O

NH

N

N

N

NH2

NH

NH

O

O

HO

O

OH

SS

H

OH

O

Folic Acid

Lipoic Acid

HN

OHS

O

NH

NH2

HO

O OH

O

Glutathione

78

Thiamin Dependent Reactions

Acetolactate synthase

CH3C

O

CO2H2thiamin

CH3C

O

C

OH

CH3

CO2H + CO2

Pyruvate dehydrogenase: pyruvate decarboxylase,

dihydrolipoyl transacetylase, dihydrolipoyl dehydrogenase,

CH3C

O

CO2H

pyruvate

+ thiamin + lipoic acid + CoA-SH Acetyl-CoA + CO2(CH3COS-CoA)

N N

S OP

-O O

O PO

-

O-

O

NH2

N

+

Thiamin Diphosphate (pyrophosphate)

SH

NH

OSH3C

O

Lys

S

NH

OLys

S

CO2-

O

- CO2

thiamin, CoA-SH

S-CoA

O

Pyruvate Decarboxylase

CH3C

O

CO2Hthiamin

CH3C

O

H + CO2

84

Pyridoxal Phosphate (PLP) dependent enzymes (Vitamin B6)

(Bugg, Chapt. 9, pp 186-198)

Involved in amino acid biosynthesis, metabolism and catabolism

N

2-O3POOH

O H

N

2-O3POOH

NH2

pyridoxal

N

2-O3POOH

OH

pyridoxamine pyridoxine

R

NH3

CO2-

H

L-amino acid

R

NH3

HCO2

-

D-amino acid

racemase,epimerase

R

NH3

HH

decarboxylase

R

O

CO2-

transaminase

R

NH2

CO2-

H2O

R'

NH3

CO2-

H

reaction at the or carbon

85

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