Enzymes chp-6-7-bioc-361-version-oct-2012b

Jody Haddow - UAEU

Chapter SixThe Behavior of Proteins: Enzymes

What are Enzymes?

• Enzymes are catalytically active biological macromolecules

• Specific, Efficient, Active in Aqueous Solution

• Most enzymes are globular proteins, however some RNA (ribozymes, and ribosomal RNA) also

catalyze reactions

Non-peptide Co-Factors (Metals, Vitamins, Coenzymes)

• Enzymes can be classified functionally

Carbonic Anhydrase

Tissues

Lungs and Kidney

107 rate enhancement

Why Biocatalysis?

• Higher reaction rates• Greater reaction specificity• Milder reaction conditions• Capacity for regulation

COO

OH

O COO

COO

O COO

NH2

OOCCOO

O

OH

OH

COO

NH2

COO

-

-

-

-

-

-

--

Chorismate mutase

• Metabolites have many potential

pathways of decomposition

• Enzymes make the desired one most

favorable

Enzymatic Substrate Selectivity

No binding

OOC NH3

H

OOC NH3

H

HNH

HOH

OH

H

OH

CH3

OOC NH3

H

OH

--

-

+

+

+

Binding but no reaction

Example: Phenylalanine hydroxylase

Enzyme Catalysis

• Enzyme: a biological catalyst• with the exception of some RNAs that

catalyze their own splicing (Section 10.4), all enzymes are proteins

• enzymes can increase the rate of a reaction by a factor of up to 1020 over an uncatalyzed reaction

• some enzymes are so specific that they catalyze the reaction of only one stereoisomer; others catalyze a family of similar reactions

• The rate of a reaction depends on its activation energy, G°‡

• an enzyme provides an alternative pathway with a lower activation energy

Enzyme Catalysis (Cont’d)

• Consider the reaction

H2O2 H2O + O2

Temperature dependence of catalysis

• Temperature can also catalyze reaction (increase rate)

• This is dangerous, why?

• Increasing temperature will eventually lead to protein denaturation

Michaelis-Menten Kinetics

• Initial rate of an enzyme-catalyzed reaction versus substrate concentration

Why Study Enzyme Kinetics?

• Quantitative description of biocatalysis• Determine the order of binding of substrates• Elucidate acid-base catalysis• Understand catalytic mechanism• Find effective inhibitors• Understand regulation of activity

Initial Rates, v0

• Linear region

• [S] [S]≅ 0

• [P] 0≅• Enzyme kinetics

saturable

• V0 = Vmax when [S]= ∞

Michaelis-Menten Model

• For an enzyme-catalyzed reaction

• The rates of formation and breakdown of ES are given by these equations

• At the steady state

rate of formation of ES = k1[E][S]

rate of breakdown of ES = k-1[ES] + k2[ES]

k1[E][S] = k-1[ES] + k2[ES]

E + S ES Pk1

k-1

k2

Michaelis-Menten Model (Cont’d)

• When the steady state is reached, the concentration of free enzyme is the total less that bound in ES

• Substituting for the concentration of free enzyme and collecting all rate constants in one term gives

• Where KM is called the Michaelis constant

[E] = [E]T - [ES]

([E]T - [ES]) [S]

[ES] k-1 + k2

k1

= = KM

Michaelis-Menten Model (Cont’d)

• It is now possible to solve for the concentration of the enzyme-substrate complex, [ES]

• Or alternatively [ES] =[E]T [S]KM + [S]

[E]T [S] - [ES][S]

[ES]= KM

= KM[ES]

[E]T [S] = [ES](KM + [S])

[E]T [S] - [ES][S]

Michaelis-Menten Model (Cont’d)

• In the initial stages, formation of product depends only on the rate of breakdown of ES

• If substrate concentration is so large that the enzyme is saturated with substrate [ES] = [E]T

• Substituting k2[E]T = Vmax into the top equation gives

Vinit = k2[ES] = k2[E]T [S]KM + [S]

Vinit = Vmax = k2[E]T

Vmax [S]Vinit = KM + [S]

Michaelis-Mentenequation

Michaelis-Menten Model (Cont’d)

• When [S]= KM, the equation reduces to

Vmax [S]V =

KM + [S]=

Vmax [S]

[S] + [S]=

Vmax

2

Linearizing The Michaelis-Menten Equation

• It is difficult to determine Vmax experimentally

• The equation for a hyperbola

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

V1 =

KM + [S]

Vmax [S]=

KM [S]Vmax [S] Vmax [S]

+

V1 =

KM

Vmax [S] Vmax

+ 1

Vmax [S]V =

KM + [S](an equation for a hyperbola)

Lineweaver-Burk Plot

• The Lineweaver-Burke plot has the form y = mx + b, and is the formula for a straight line

• a plot of 1/V versus 1/[S] will give a straight line with slope of KM/Vmax and y intercept of 1/Vmax

• such a plot is known as a Lineweaver-Burk double reciprocal Lineweaver-Burk double reciprocal plotplot

V1 =

Vmax

+ 1Vmax [S]

1

y m x + b

V1 =

KM •

= •

Lineweaver-Burk Plot (Cont’d)

• KM is the dissociation constant for ES; the greater the value of KM, the less tightly S is bound to E

• Vmax is the turnover number

Turnover Numbers

• Vmax is related to the turnover number of enzyme:also called kcat

• Number of moles of substrate that react to form product per mole of enzyme per unit of time

V max

[ET ]

turnover _ number kcat

Chapter SevenThe Behavior of Proteins:

Enzymes, Mechanisms, and Control

Enzymes fall into classes based on function

• There are 6 major classes of enzymes:

1.Oxidoreductases which are involved in oxidation, reduction, and electron or proton transfer reactions;

2.Transferases, catalysing reactions in which groups are transferred;

3.Hydrolases which cleave various covalent bonds by hydrolysis; 4

4.Lyases catalyse reactions forming or breaking double bonds;

5.Isomerases catalyse isomerisation reactions;

6.Ligases join substituents together covalently.

Allosteric Enzymes

• Allosteric:Allosteric: Greek allo + steric, other shape• Allosteric enzymeAllosteric enzyme:: an oligomer whose biological activity is affected by

other substances binding to it

• these substances change the enzyme’s activity by altering the conformation(s) of its 4°structure

• Allosteric effectorAllosteric effector:: a substance that modifies the behavior of an allosteric enzyme; may be an

• allosteric inhibitor• allosteric activator

• Aspartate transcarbamoylase (ATCase)Aspartate transcarbamoylase (ATCase)

• feedback inhibitionfeedback inhibition

Feedback Inhibition

Formation of product inhibits its continued production

Allosteric Regulation; ATCase

Enzyme Inhibition

Inhibitors are compounds that decrease enzyme’s activity

• Irreversible inhibitors (inactivators) react with the enzyme- one inhibitor molecule can permanently shut off one enzyme molecule

- they are often powerful toxins but also may be used as drugs

• Reversible inhibitors bind to, and can dissociate from the enzyme - they are often structural analogs of substrates or products

- they are often used as drugs to slow down a specific enzyme

• Reversible inhibitor can bind: – To the free enzyme and prevent the binding of the

substrate

– To the enzyme-substrate complex and prevent the

reaction

Types of Inhibition

• Competitive Inhibition• Noncompetitive Inhibition• Irreversible Inhibition

.

S

I

Enzyme

Competitive Inhibition

In competitive inhibition, the inhibitor competes with the substrate for the same binding site

Competitive inhibitors

• Enzymes can be inhibited competitively, when the substrate and inhibitor compete for binding to the same active site

• This can determined by plotting enzyme activity with and without the inhibitor present.

• Competitive Inhibition• In the presence of a competitive inhibitor, it takes a

higher substrate concentration to achieve the same velocities that

• were reached in its absence. So while Vmax can still be reached if sufficient substrate is available, one-half Vmax requires a higher [S] than before and thus Km is larger.

Competitive Inhibition - Reaction Mechanism

In competitive inhibition, the inhibitor binds only to the free enzyme, not to the ES complex

E + S ES E + P

EI

+I

General Michaelis-Menten Equation

This form of the Michaelis-Menten equation can be used to understand how each type of inhibitor affects the reaction rate curve

v =[S]

Km,app + [S]Vmax,app

In competitive inhibition, only the apparent Km is affected (Km,app> Km),

The Vmax remains unchanged by the presence of the inhibitor.

Competitive inhibitors alter the apparent Km, not the Vmax

.

Vmax

Vmax

2

Km Km,app

[Substrate]

Rea

ctio

n R

ate

- Inhibitor

+ Inhibitor

Vmax,app = Vmax

Km,app > Km

The Lineweaver-Burk plot is diagnostic for competitive inhibition

Slope =Km,app

Vmax

1Vmax

-1Km,app

1[S]

Increasing [I]

1v

v=1

Vmax

Km,app

Vmax

1+[S]1

.

Vmax

Vmax

2

Km Km,app

[Substrate]

Rea

ctio

n R

ate

- Inhibitor

+ Inhibitor

.

S

I

Inhibitor competes with

substrate, decreasing its

apparent affinity: Km,app > Km

Formation of EIcomplex shifts reactionto the left: Km,app > Km

Km,app > Km

Vmax,app = VmaxE + S ES E + P

EI

+I

Formation of EIcomplex shifts reaction

to the left: Km,app > Km

Relating the Michaelis-Menten equation, the v vs. [S] plot, and the physical picture of competitive inhibition

.

S

I

IS

S

I

I

S

Enzyme

Enzyme

Enzyme

Enzyme

Noncompetitive Inhibition

the inhibitor does not interfere with substrate binding (and vice versa)

Non-competitive inhibitor

• With noncompetitive inhibition, enzyme molecules that have been bound by the inhibitor are taken out of the game so enzyme rate (velocity) is reduced for all values of [S], including Vmax and one-half Vmax but

• Km remains unchanged because the active site of those enzyme molecules that have not been inhibited is unchanged.

E + S ES E + P

EI

+I

ESI

+I

S+

Noncompetitive Inhibition - Reaction Mechanism

In noncompetitive inhibition, the inhibitor binds enzyme irregardless of whether the substrate is bound

Noncompetitive inhibitors decrease the Vmax,app, but don’t affect the Km

Vmax,app < Vmax

Km,app = Km

.

Vmax

Vmax21

21 Vmax,app

Km

Km,app

[Substrate]

Re

ac

tio

n R

ate

- Inhibitor

+ InhibitorVmax,app

The inhibitor binds equally well to free enzyme and the ES

complex, so it doesn’t alter apparent affinity of the enzyme for the

substrate

Why does Km,app = Km for noncompetitive inhibition?

E + S ES E + P

EI

+I

ESI

+I

S+

The Lineweaver-Burk plot is diagnostic for noncompetitive inhibition

v=1

Vmax,app

Km

Vmax,app

1+[S]1

Slope =Vmax,app

Km

1Vmax,app

-1Km

1[S]

Increasing [I]

1v

Formation of EIcomplex shifts reactionto the left: Km,app > Km

Km,app > Km

Vmax,app = Vmax

.

S

I

IS

S

I

I

S

Enzyme

Enzyme

Enzyme

Enzyme

.

Vmax

Vmax21

21 Vmax,app

Km Km,app

[Substrate]

Re

ac

tio

n R

ate

- Inhibitor

+ InhibitorVmax,app

Inhibitor doesn’t interferewith substrate binding,

Km,app = Km

E + S ES E + P

EI

+I

ESI

+I

S+

Even at highsubstrate levels,

inhibitor still binds,

[E]t < [ES]Vmax,app < Vmax

Vmax,app < Vmax

Km,app = Km

Relating the Michaelis-Menten equation, the v vs. [S] plot, and the physical picture of noncompetitive inhibition

Irreversible Inhibition

In irreversible inhibition, the inhibitor binds to the enzyme irreversibly through formation of a covalent bond with the enzyme , permanently inactivating the enzyme

.

Enzyme

SO I

Irreversible Inhibition - Reaction Mechanism

In irreversible inhibition, the inhibitor permanently inactivates the enzyme. The net effect is to remove enzyme from the reaction. Vmax decreases

No effect on Km

E + S ES E + P

EI

+I

The Michaelis-Menten plot for an irreversible inhibitor looks like noncompetitive inhibition

Vmax,app < Vmax

Km,app = Km

.

Vmax

Vmax21

21 Vmax,app

Km

Km,app

[Substrate]

Re

ac

tio

n R

ate

- Inhibitor

+ InhibitorVmax,app

Irreversible inhibition is distinguished from noncompetitive inhibition by plotting Vmax vs [E]t

Enzyme is inactivated until all of the irreversible inhibitor is used up

.

[E]t

Vm

ax

+ Reversible

Noncompetitive In

hibitor

- Inh

ibito

r

+ Ir

reve

rsib

le In

hibi

tor[E]t > [I][E]t < [I]

[E]t = [I]

Summary-Enzyme Inhibition

• Competitive Inhibitor• Binds to substrate binding site • Competes with substrate • The affinity of the substrate appears to be decreased

when inhibitor is present (Km,app >Km)

• Noncompetitive inhibitor• Binds to allosteric site• Does not compete with the substrate for binding to the

enzyme• The maximum velocity appears to be decreased in the

presence of the inhibitor (Vmax,app <Vmax)

• Irreversible Inhibitor• Covalently modifies and permanently inactivates the

enzyme

Competitive/noncompetitive inhibitor

Effect of inhibitors

Enzyme Regulation

• Allosteric regulation, • heterotropic ligand binding modulates

substrate binding and catalysis, • Feedback regulates metabolic pathways

• Covalent modification – Reversible • Phosphorylation, nucleotides, lipid anchors

• Proteolysis converts inactive pro-enzymes (zymogens) to active

Allosteric Enzymes

• Effector molecules change the activity of an enzyme by binding at a second site• Some effectors speed up enzyme action (positive

allosterism)

• Some effectors slow enzyme action (negative allosterism)

Protein Modification

• In protein modification a chemical group is covalently added to or removed from the protein• Covalent modification either activates or turns off the

enzyme

• The most common form of protein modification is addition or removal of a phosphate group• This group is located at the R group (with a free –

OH) of:• Serine • Threonine• Tyrosine

Control of Enzyme Activity via 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

• Membrane transport is a common example

Covalent ModificationLipase:

Proenzymes

• A proenzyme, an enzyme made in an inactive form

• It is converted to its active form• By proteolysis (hydrolysis of the enzyme)• When needed at the active site in the cell• Pepsinogen is synthesized and transported to the

stomach where it is converted to pepsin

Coenzymes

• Coenzyme:Coenzyme: a nonprotein substance that takes part in an enzymatic reaction and is regenerated for further reaction• metal ions- can behave as

coordination compounds. (Zn2+, Fe2+)

• organic compounds, many of which are vitamins or are metabolically related to vitamins (Table 7.1).

NAD+/NADH

• Nicotinamide adenine dinucleotide (NAD+) is used in many redox reactions in biology.

• Contains:

1) nicotinamide ring

2) Adenine ring

3) 2 sugar-phosphate groups

NAD+/NADH (Cont’d)

• NAD+ is a two-electron oxidizing agent, and is reduced to NADH

• Nicotinamide ring is where reduction-oxidation occurs