Enzyme

Course: B.Sc.(Biochem) II

Subject: Biomolecules I

Unit 4

Enzymes

What are enzymes?

Enzymes are complex chemicals that control reactions in living cells. They are

biochemical catalysts speeding up reactions that would otherwise happen too slowly.

The chemical which an enzyme works on is called its substrate.

An enzyme combines with its substrate to form a short-lived enzyme/substrate complex.

Once a reaction has occurred, the complex breaks up into products and enzyme.

E + S ES EP E + P

The enzyme remains unchanged at the end of reaction and is free to interact again with

more substrate.

How the confectioners make the runny yolk-like

inside to chocolate eggs?

The answer is : use of an enzyme

The role of enzymes in an organism

Many of the complex chemicals that living organisms need cannot be made in a single

reaction. Instead a series of simpler reactions occur, one after another, forming a metabolic

pathway. A single pathway may have many steps in which each chemical is converted to

the next. A specific enzyme controls each reaction.

Enzymes control cell metabolism by regulating how and when reactions occur. Using

this very simple pathway as an example:

A B C D

The final product is substance D, the chemical needed by the living organism. The

pathway needs three different enzymes and when D is no longer needed or if too much has

been produced, one of the three enzymes is ‘switched off’.

1

The Chemical nature of enzymes

Enzymes are globular proteins. They have a complex tertiary and quaternary structure

in which polypeptides are folded around each other to form a roughly spherical or globular

shape. The overall 3D shape of an enzyme molecule is very important: if it is altered, the

enzyme cannot bind to its substrate and so cannot function. Enzyme shape is maintained

by hydrogen bonds and ionic forces.

Enzymes have several important properties:

Enzymes are specific: each enzyme usually catalyses only one reaction.

Enzymes combine with their substrates to form temporary enzyme-substrate complex.

Enzymes are not altered or used up by the reactions they catalyze, so can be used again

and again.

Enzymes are sensitive to temperature and pH.

Many enzymes need cofactors in order to function.

Enzyme function may be slowed down or stopped by inhibitors.

Characteristics of Enzymes?Enzymes

1)speed up chemical reactions

2)are required in minute amounts

3)are highly specific in their action

4)are affected by temperature

5)are affected by pH

6)Some catalyse reversible reactions

7)Some require co-enzymes

8)Are inhibited by inhibitors

(1) Enzymes Speed up chemical reactions

Activation Energy

with enzyme

Activation

Energy without

enzyme

Substrate

Products:

Energy

Time

• By lowering the activation energy needed to start

the reaction.

(2) Enzymes are required in minute amounts

Sucrose Glucose + Fructose

• They remain chemically unchanged after catalysing

the reactions.

• The same enzyme molecules can be reused over

again.

• Therefore, only a small amount of enzyme is required

to catalyse a large number of reactions

Sucrase

Chemically unchanged

Maltose Glucose + Glucose

(3) Enzymes are highly specific

• Each chemical reaction is catalysed by its own specific,

unique enzyme.

• This is due to every enzyme’s specific 3-d configuration.

• How the shape of an enzyme affects its function can be

explained by the “LOCK & KEY HYPOTHESIS”.

Maltase

Starch MaltoseAmylase

Enzyme Active SitesUnder biologically relevant conditions, uncatalyzed reactions tend to be slow because most biological molecules are quite stable in the neutral-pH, mild-temperature, aqueous environment inside cells. Enzymes greatly increase the rates of biological reactions by providing a specific environment within which a reaction can occur more rapidly. Enzyme-catalyzed reactions take place within the confines of a pocket on the enzyme called the active site. The reactant molecule is referred to as the substrate. The surface of the active site is lined with amino acid residues with substituent groups that bind to the substrate and catalyze its chemical transformation. Often, the active site encloses the substrate, sequestering it from solution. The active site of the enzyme chymotrypsin is highlighted in Fig. 6-1.

2

ENZYME POWER!

All reactants need to have a certain energy before they will react. This is like an energy barrier that it has to overcome before a reaction will occur. It is called the activation energy.

Enzymes are organic catalysts.

All catalysts lower the energy barrier, allowing the reactants (substrates) to react faster forming the products.

Enzymes do not participate in the reaction.

Reactant

Product

Without enzyme: The activation

energy required is high.

With enzyme: The activation

energy required is lower.

ENZYMES

High

Low

Start Finish

Direction of reaction

Am

ou

nt o

f e

ne

rgy s

tore

d in

th

e

ch

em

ica

ls

Low energy

High energy

ENZYMES

Enzymes have a specific region where

the substrate binds and where catalysis

occurs. This is called the active site.

Enzymes are substrate-specific,

although specificity varies from enzyme

to enzyme.

When a substrate binds to an enzyme’s

active site, an enzyme-substrate

complex is formed. Space filling model of the yeast

enzyme hexokinase. Its active

site lies in the groove (arrowed)

3

LOCK AND KEY MODEL

The lock and key model of enzyme action, proposed earlier this century,

proposed that the substrate was simply drawn into a closely matching cleft

on the enzyme molecule.

Substrate

Enzyme

Products

Symbolic representation of the lock and key model of enzyme action.

1. A substrate is drawn into the active sites of the enzyme.

2. The substrate shape must be compatible with the enzymes active site in

order to fit and be reacted upon.

3. The enzyme modifies the substrate. In this instance the substrate is

broken down, releasing two products.

4

INDUCED FIT MODEL

More recent studies have

revealed that the process is much

more likely to involve an

induced fit.

The enzyme or the reactants

(substrate) change their shape

slightly.

The reactants become bound to

enzymes by weak chemical bonds.

This binding can weaken bonds

within the reactants themselves,

allowing the reaction to proceed

more readily.

The enzyme

changes shape,

forcing the substrate

molecules to

combine.

Two substrate

molecules are

drawn into the cleft

of the enzyme.

The resulting end

product is released

by the enzyme

which returns to its

normal shape, ready

to undergo more

reactions.

5

CHANGING THE ACTIVE SITE

Changes to the shape of the active site will result in a loss of

function. Enzymes are sensitive to various factors such as

temperature & pH.

When an enzyme has lost its characteristic 3D shape, it is said to

be denatured. Some enzymes can regain their shape while in

others, the changes are irreversible.

THE EFFECT OF TEMPERATURE ON ENZYME

ACTION Speeds up all reactions, but the

rate of denaturation of enzymes also increases at higher temperatures.

High temperatures break the disulphide bonds holding the tertiary structure of the enzyme together thus changing the shape of the enzyme.

This destroys the active sites & therefore makes the enzyme non – functional.

Too cold for Enzyme to

work

Too hot for Enzyme to

work

Optimum Temperature for enzyme

THE EFFECT OF TEMPERATURE ON ENZYME

ACTION

The curve in the blue represents an enzyme isolated from an organism living in the artic. These cold dwelling organisms are called psychrophiles.

The curve in red represents an enzyme isolated from the digestive tract of humans.

The curve in green represents an enzyme isolated from a thermophile bacteria found growing in geothermal sea beds.

THE EFFECT OF PH ON ENZYME ACTION

Like all proteins, enzymes are denatured by extremes of pH (acidity/alkalinity).

The green curve is for pepsin that digests proteins in the stomach.

The red curve represents the activity of arginase that breaks down arginine to ornithine & urea in the liver.

THE EFFECT OF ENZYME

CONCENTRATION ON ENZYME ACTION

Assuming that the

amount of substrate is

not limiting, an increase

in enzyme concentration

causes an increase in the

reaction rate.

THE EFFECT OF SUBSTRATE

CONCENTRATION ON ENZYME ACTION

Assuming that the amount of enzyme is constant, an increase in substrate concentration causes a diminishing increase in the reaction rate.

A maximum rate is obtained at a certain concentration of substrate when all enzymes are occupied substrate (the rate cannot increase any further).

THE EFFECT OF COFACTORS ON ENZYME ACTION

Cofactors are substances that are essential to the catalytic activity of some enzymes.

Cofactors may alter the shape of enzymes slightly to make the active sites functional or to complete the reactive site.

Enzyme cofactors include coenzymes (organic molecules) or activating ions (eg. Na+, K+..)

Vitamins are often coenzymes (eg. Vit B1, Vit B6…)

6

THE NATURE OF ENZYME INHIBITORS

Enzyme inhibitors may or may not act reversibly:

Reversible: the inhibitor is temporarily bound to the enzyme, thereby preventing its function (used as a mechanism to control enzyme activity).

Irreversible: the inhibitor may bind permanently to the enzyme causing it to be permanently deactivated.

THE NATURE OF ENZYME INHIBITORS

Reversible Enzymes work in one of two ways:

Competitive inhibitors: the inhibitor competes with the substrate for the active site, thereby blocking it and preventing attachment of the substrate.

Non-competitive: the inhibitor binds to the enzyme (but not at the active site) and alters its shape. It markedly slows down the reaction rate by making the enzyme less able to perform its function (allosteric inhibition).

The Role of the ES Complex (I)The ES complex is the key to understanding the kinetic behavior of an enzyme. In 1913, Leonor Michaelis and Maud Menten, developed a kinetic equation to explain the behavior of many simple enzymes. Key to the development of their equation, is the assumption that the enzyme first combines with its substrate to form an enzyme-substrate complex in a relatively fast reversible step:

k1

E + S ES

k-1

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

k2

ES E + P

k-2

If the slower second reaction limits the rate of the overall reaction, the overall rate must be proportional to the concentration of the species that reacts in the second step, i.e., ES.

At any given instant in an enzyme-catalyzed reaction, the enzyme exists in two forms, the free or uncombined form E and the combined form ES. At low [S], most of the enzyme is in the uncombined E form. Here, the rate is proportional to [S] because the direction of the first equation above is pushed toward formation of more ES as [S] increases. (Continued on the next slide).

The Role of the ES Complex (II)The maximum initial rate of the catalyzed reaction (Vmax) is observed when virtually all of the enzyme is present in the ES complex and [E] is vanishingly small. Under these conditions, the enzyme is saturated with its substrate, so that further increases in [S] have no effect on rate. This condition exists when [S] is sufficiently high that essentially all the free enzyme has been converted to the ES form. The saturation effect is a distinguishing characteristic of enzymatic catalysts and is responsible for the plateau observed in Fig. 6-11. The pattern seen in that figure is sometimes referred to as saturation kinetics.

When the enzyme is first mixed with a large excess of substrate, there is an initial period, the pre-steady state, during which the concentration of ES builds up. This period is usually too short to be easily observed, lasting just microseconds, and is not evident in Fig. 6-10. The reaction quickly achieves a steady state in which [ES] remains approximately constant over time. The measured V0 generally reflects the steady state, even though V0 is limited to the early part of the reaction. The analysis of these initial rates is referred to as steady-state kinetics.

Derivation of the MM Equation (I)The kinetic curves expressing the relationship between V0 and [S] have the same general shape (a rectangular hyperbola) for most enzymes, which can be expressed algebraically by the MM equation. Michaelis and Menten derived this equation starting from their basic hypothesis that the rate-limiting step in enzymatic reactions is the breakdown of the ES complex to product and free enzyme. The MM equation is

V0 = Vmax[S]/(Km + [S]).

All these terms, [S], V0, Vmax, as well as the constant called the Michaelis constant, Km, can be readily measured experimentally.

The derivation of the MM equation starts with the two basic steps of the formation and breakdown of ES. Early in the reaction, the concentration of the product [P] is negligible, and a simplifying assumption is made that the reaction P S (described by k-2) can be ignored. The overall reaction then reduces to

k1 k2

E + S ES E + P.

k-1

Derivation of the MM Equation (II)V0 is determined by the breakdown of ES to form product, which is determined by [ES] through the equation

V0 = k2[ES].

Because [ES] in the above equation is not easily measured experimentally, an alternative

expression for this term must be found. First, the term [Et], representing the total enzyme

concentration (the sum of free and substrate-bound enzyme) is introduced. Free or unbound

enzyme [E] can then be represented by [Et] - [ES]. Also, because [S] is ordinarily far

greater than [Et], the amount of substrate bound by the enzyme at any given time is

negligible compared with the total [S]. With these conditions in mind, the following steps

lead to an expression for V0 in terms of easily measurable parameters.

Derivation of the MM Equation (III)

Derivation of the MM Equation (IV)

Derivation of the MM Equation (V)

The MM equation describes the kinetic behavior of a great many enzymes, and all enzymes that exhibit a hyperbolic dependence of V0 on [S] are said to follow Michaelis-Menten kinetics. However the MM equation does not depend on the relatively simple two-step reaction mechanism discussed above. Many enzymes that follow MM kinetics have quite different mechanisms, and enzymes that catalyze reactions with six or eight identifiable steps often exhibit the same steady-state kinetic behavior. Even though the MM equation holds true for many enzymes, both the magnitude and the real meaning of Vmax and Km can differ from one enzyme to another. This is an important limitation of the steady-state approach to enzyme kinetics.

Validation of the MM EquationThe MM equation can be shown to correctly explain the V0 vs [S] curves of many enzymes by considering limiting situations where [S] is very high or very low (Fig. 6-12). At low [S], Km >> [S] and the [S] term in the denominator of the MM equation becomes insignificant. The equation simplifies to V0 = Vmax[S]/Km and V0 exhibits a linear dependence on [S], as is observed at the left side of V0 vs [S] graphs. At high [S], where [S] >> Km, the Km term in the denominator of the MM equation becomes insignificant and the equation simplifies to V0 = Vmax. This is consistent with the plateau in V0 observed at high [S] in kinetic graphs.

An important numerical relationship emerges from the MM equation in the special case when V0 is exactly one-half Vmax. Here

Vmax/2 = Vmax[S]/(Km + [S]).

On dividing by Vmax, the equation is

1/2 = [S]/(Km + [S]).

After solving for Km, we get

Km + [S] = 2[S], or Km = [S].

This is a very useful, practical definition of Km. Km is equivalent to the substrate concentration at which V0 is one-half Vmax.

Double-reciprocal PlotsBecause the plot of V0 vs [S] for an enzyme-catalyzed reaction asymptotically approaches the value of Vmax at high [S], it is difficult to accurately determine Vmax (and thereby, Km) from such graphs. The problem is readily solved by converting the Michaelis-Menten kinetic equation to the so-called double-reciprocal equation (Lineweaver-Burk equation) which describes a linear plot from which Vmax and Km can be easily obtained (Box 6-1, Fig. 1). The Lineweaver-Burk equation is derived by first taking the reciprocal of both sides of the Michaelis-Menten equation

1/V0 = (Km + [S])/Vmax[S]

Separating the components of the numerator on the right side of the equation gives

1/V0 = Km/Vmax[S] + [S]/Vmax[S]

Which simplifies to

1/V0 = Km/Vmax[S] + 1/Vmax.

The plot of 1/V0 vs 1/[S] gives a straight line, the y-intercept of which is 1/Vmax and the x-intercept of which is -1/Km.

The Meaning of the KmThe Km can vary greatly from enzyme to enzyme, and even for different substrates of the same enzyme (Table 6-6). The Km is sometimes used (often inappropriately) as an indicator of the affinity of an enzyme for its substrate. The actual meaning of the Kmdepends on specific aspects of the reaction mechanism such as the number and relative rates of the individual steps. For example, for a reaction with two steps, Km = (k2 + k-1)/k1. If k2 is actually rate-limiting, then k2 << k-1, and Km reduces to k-1/k1, which is the dissociation constant, Kd of the ES complex. Where these conditions hold, Km does represent a measure of the affinity of the enzyme for its substrate. However this scenario often doesn’t apply for an enzyme due to variances in the contributions of individual rate constants to the overall reaction rate. Thus Km cannot always be considered a simple measure of the affinity of an enzyme for its substrate.

Information Derived from VmaxThe meaning of the quantity Vmax also varies greatly from one enzyme to the next. If an enzyme reacts via the two-step MM mechanism, then Vmax = k2[Et]. However, the number of reaction steps and the identity of the rate-limiting step can vary from enzyme to enzyme. Therefore, it is useful to define a more general rate constant, kcat, to describe the rate constant of the rate limiting step(s) of any enzyme-catalyzed reaction at saturation. With the modification that Vmax = kcat[Et], the MM equation becomes

V0 = kcat[Et][S]/(Km + [S]).

The constant kcat is a first-order rate constant and hence has the units of reciprocal time (s-1). It is also called the turnover number for the enzyme-catalyzed reaction. It is equivalent to the number of substrate molecules converted to product in a given unit of time on a single enzyme molecule when the enzyme is saturated with substrate. The turnover numbers of several enzymes are listed in Table 6-7.

The Specificity Constant (kcat/Km)Together, the parameters kcat and Km can be used to evaluate the catalytic efficiency of an enzyme. The best way to determine the catalytic efficiency of an enzyme is to determine the ratio of kcat/Km for its reaction. This parameter, sometimes called the specificity constant, is the rate constant for the conversion of E + S to E + P. When [S] << Km, the previous MM equation form converts to

V0 = (kcat/Km)([Et][S]).

V0 in this case depends on the concentration of two reactants, [Et] and [S]. Therefore, this is a second-order rate equation and the constant kcat/Km is a second-order rate constant with the units M-1s-1. There is an upper limit to kcat/Km, imposed by the rate at which E and S can diffuse together in an aqueous solution. This diffusion-controlled limit is 108 to 109 M-1s-1, and many enzymes have a kcat/Km near this range (Table 6-8). Such enzymes are said to have achieved catalytic perfection.

REFERENCES:

Image 1: http://postimg.org/image/hzxi8jlzz/

Image 2: http://postimg.org/image/97gjkv0v3/

Image 3: http://postimg.org/image/cfpmbncin/

Image 4 & 5: Harper’s Illustrated Biochemistry by Robert,

Murray, Hranner, Mayes and Rodwell

Image 6: http://postimg.org/image/k5rt31pgf/

Books:

1. Biochemistry, U. Satyanarayan, Elsevier India publication

2. Principles of Biochemistry by Lehninger