Enzymes

Bacterial Restriction endonucleases

What are enzymes?

enzymes are biological catalysts

usually proteins but RNA can also act as enzymes under specific conditions as well

catalysts are compounds that increase the rate of reactions without being
consumed in the reaction

the rate of chemical reactions depend upon the concentrations of participants

(reactants and products) and the activation energy required for a reaction to
occur

in biological systems, the concentrations of reactants are usually very low and
significant activation energy barriers exist. Therefore, most biochemical
reactions would occur too slowly to permit life in the absence of catalysts.

enzymes increase the rate of reactions by addressing low reactant concentrations

and high activation energies

Enzymes do not alter reaction equilibria

An enzyme cannot alter the laws of thermodynamics and consequently cannot
alter the equilibrium of a chemical reaction Stryer
consider the reaction of Substrate (S) to Product (P):

Note that with or without

enzyme present the same
amount of product will
be formed in a chemical
reaction.
With enzyme the reaction
may take seconds and
without may take hours
(or seconds vs centuries
depending on the reaction).

Note that in the enzyme catalyzed reaction, the rate of product formation levels
off with time. This is because the reaction has reached equilibrium: substrate
is still being converted to product but product is also being converted into substrate
at a rate such that the product remains constant.

suppose in a chemical reaction that:

the rate of product formation (k1) = 1 x 10-4 mol/s
and
the rate of substrate formation (k-1) = 1 x 10-6 mol/s
10-4 s-1

10-6 s-1

The equilibrium constant for the reaction is given by the ratio of the rate constants

[P]
[S]

10-4
10-6

100

in other words, when the reaction is at equilibrium the ratio of product to substrate
is 100:1
the presence of an enzyme catalyzing the reaction will not alter this ratio (equilibrium).
but it may reduce the time to reach this equilibrium from hours or centuries to seconds
or a fraction of second

many reactions take hours, years, or thousands of years to proceed to completion

(equilibrium) but enzymes can increase rates millions of times

orotidine monophosphate
conversion to
uridine monophosphate
by
OMP decarboxylase
half-life of OMP in absence of enzyme = 78 million years
half-life of OMP in presence of enzyme = 18 milliseconds

OMP decarboxylase reaction mechanism

"OMPDC Carbanion Mechanism" by Shareef164 - I Michael created this work entirely by myself.. Licensed under CC BYSA 3.0 via Commons https://commons.wikimedia.org/wiki/File:OMPDC_Carbanion_Mechanism.png#/media/File:OMPDC_Carbanion_Mechan
ism.png

Enzymes increase the Rate of Reactions by decreasing activation energy

many of the reactions that occur in cells will occur spontaneously (-G) but occur
too slowly for life processes

consider the oxidation of glucose

C6H12O6 + 6 O2 6 CO2 + 6 H2O

the G is about -2872 kJ/mol and a large amount of heat is liberated in this
spontaneous reaction.
The equilibrium for this reaction is far on the right side of the above equation
because thermodynamically, the products are less energetic and therefore more stable
and less ordered.
The presence of glucose is not favored in this universe (at least in the presence of
oxygen) yet a bottle of glucose can sit on a shelf for hundreds of years and not
become substantially oxidized.

 An enzyme can quickly bring about the oxidation of glucose however

by lowering the energy barrier to the forward (oxidation) reaction

Note that a spontaneous reaction will proceed where the products have less
energy than the reactants (substrate) and G is the free energy change
between the product and the reactant.

Note that the enzyme does not alter the free energy change in a reaction which
is why it does not alter the equilibrium associated with a reaction - only the rate.

Enzymes lower Activation Energy by stabilizing a Transition State

intermediate
the free energy difference between reactants and products determines the
equilibrium for a reaction
but enzymes determine how quickly the equilibrium is reached by helping to
form a transition state chemical form that is no longer reactant (substrate) but
not yet product in structural terms

where denotes transition state

The transition state is a high energy and unstable

intermediate that must form during the conversion
of substrate to product. It has a very short half-life
(about 1 x 10-13 s).
Its high energy is the source of activation energy
(G).
Enzymes therefore lower activation energy by providing
a chemical scaffold that facilitates the formation of
the transition state molecule

Enzymes therefore lower activation energy by providing

a chemical scaffold that facilitates the formation of
the transition state molecule

when two chemicals react, the ability to form a transition state intermediate
and eventual product relies on two main conditions:

the kinetic energy of the colliding substrate molecules

the orientation of the colliding substrate molecules

adding an external source of heat energy is one way to provide the required
kinetic energy to substrate molecules such that they are able to form
the transition state intermediate
an enzyme reduces activation energy by binding two reactants, bringing
them physically together in a orientation that permits reaction.

This occurs in the ACTIVE SITE of the enzyme.

Substrates bind in the active site of an enzyme

a relatively small portion of an enzyme is involved in catalysis

the active site accommodates only specific substrates on the basis of size, shape,
and chemical characteristics of the substrate

the induced fit model describes that both the structure of the enzyme and the
substrate is changed upon binding

Amino acid side-chains interact with substrate molecules through weak forces
such as ionic, hydrogen-bonding, and van der Waals interactions.

it is these inter-molecular interactions that determine a) binding specificity,

b) the orientation of substrate molecules in the active site, c) stabilization of
the transition state structure, d) chemical reactivity or catalysis

Catalytic amino acids reside in the active site

the active site binding pocket or cleft is where several amino acid side chains that
play instrumental roles in chemical catalysis coincide

these are called active site residues or catalytic residues

Naturally, the folding of a

protein into its tertiary state
is not a random process.

Protein Evolution

as species diverge from one another, mutations will inevitably and independently
occur in the genes that encode proteins.

some mutations may be lethal and the host organism dies

other mutations are permitted, leading to divergence amongst the amino acid
sequences of like-proteins from different species.

Here are four like-proteins from different bacterial species.

These four proteins carry out the same function in each bacterium.
1
2
3
4

N
N
N
N

MYFGHATFRVVARFPLKMNKYTWYHCNPCKACIYTGDEGGAKIADENS
MDKWAKWFMVAARGLCADDKLAQRHVQLCQACKMAADELNWEIALPAV
MSVKLGCWQVLARDEDSTGKAFERHVEICQACKLMADEAGSEIAYCVV
MDEWAVNFMVVARHLAADQRLANRHVADCQACKAWADDKQMEIVFQAV

Note that not all amino acids have similar importance in the protein. Some are
important for structure (pink) and some are active site catalytic residues (blue) and
these are largely conserved.
Some amino acids at specific positions are not conserved at all, indicating they
are not overtly important to the protein or enzyme

C
C
C
C

Some Enzymes possess co-factors required for either structure

or catalysis

some enzymes function on their own while some have co-factors intimately
associated with their structure

co-factors may be metal ions or larger molecules called co-enzymes

both of these often participate in the catalysis process

co-enzymes are often vitamin derivatives

A Holoenzyme forms
when the Apoenzyme form
of the protein binds its
co-factor

Some selected co-factors (coenzymes and metal ions)

for enzymes that require co-factors, the protein lacking the co-factor is inactive
this is the central reason why vitamins and certain metal ions are essential for life

Thiamine, Vit B1
Riboflavin, Vit B2
Niacin
Vitamin B6
Pantothenic acid
Vit B12
Folic acid

the 5 essential ions

Ca2+, K+, Na+, Mg2+, Cl-

The catalytically important amino acids and the co-factors define the active site
of the enzyme and interact with substrate, the transition state intermediate
and products formed

Active site amino acid side chains of the enzyme Cytochrome P450
and its co-factor Heme interacting with its substrate camphor

Active site amino acid side chains specifically interact with substrate molecules
as shown in the active site of Ribonuclease (an enzyme that hydrolyzes RNA
molecules to free ribonucleotides).

We will talk about the

mechanism by which
enzymes catalyze
chemical reaction later

Next few lectures we will cover:

Enzyme kinetics (the study of the rate of enzyme catalysis)

Types of catalysis
The Chemical Mechanisms of enzymatic catalysis