Introduction to Molecular Cell Biology

Biocatalysts Biocatalysts Dr. Fridoon Jawad AhmadDr. Fridoon Jawad Ahmad

HEC Foreign ProfessorHEC Foreign ProfessorKing Edward Medical UniversityKing Edward Medical University

Visiting Professor LUMS-SSEVisiting Professor LUMS-SSE

The Miracle Workers

One of biggest miracles of life is that it can perform slow reactions and the ones requiring extreme

conditions, quickly and at physiological conditions.

N2(g) + 3H2(g) 2NH3(g)

N2 + 8H+ + 8e− + 16 ATP 2NH3 + H2 + 16ADP + 16 Pi

The reaction is carried out under conditions of 150-250 atmospheres (atm), 450-500 °C;

resulting in a yield of 10-20%

Nitrogenase

Energy and Energy Conversions

Energy is the capacity to do work. Potential energy is the energy of state or position; it includes the

energy stored in chemical bonds. Kinetic energy is the energy of motion (and related forms such as

electric energy, light, and heat).

The Laws of Thermodynamics

Living things, obey the laws of thermodynamics.

1) Energy cannot be created or destroyed.

2) The quantity of energy available to

do work (free energy) decreases

and unusable energy (associated

with entropy) increases.

Free Energy (ΔG)

total energy = usable energy + unusable energyH = G + TS

(H = Enthalpy, G = free energy, T= absolute temperature, S = entropy)

G = H – TS

Absolute G, H or S can not be measuredΔG (reaction) = G (products) – G (reactants)

Changes in free energy, total energy, temperature, and entropy are related by the equation.

ΔG = ΔH – TΔS

ΔG of Combustion

CH4 + 2O2 CO2 + 2H2OH = G + TS H = G + TS

500 = 400 + 100 300 = 100 + 200

G = H – TS G = H – TS400 = 500 - 100 100 = 300 - 200

ΔG (reaction) = G (products) – G (reactants)- 300 = 100 - 400

ΔG = - 300ΔH = - 200

ΔG of Reverse Reaction

CO2 + 2H2O CH4 + 2O2H = G + TS H = G + TS

300 = 100 + 200 500 = 400 + 100

G = H – TS G = H – TS100 = 300 - 200 400 = 500 - 100

ΔG (reaction) = G (products) – G (reactants) 300 = 400 - 100

ΔG = 300ΔH = 200

ΔS = - 100

Free Energy (ΔG)

If ΔG is negative free energy is releasedIf ΔG is positive free energy is required

ΔH is the total amount of energy added

In living systems magnitude and sign of ΔG can depend significantly on changes in entropy ΔS

ΔS will be positive in hydrolysis

Water Molecules adjacent to hydrophobic molecule suffer restrictions in orientation as they form hydrogen bonds with other water molecules.

Bonding (hydrogen) also reduces energy state.

Hydrophobicity Counterintuitive ???

Water Structure

Exergonic & Endergonic Reactions

Exergonic reactions release free energy and have a negative ΔG.

Endergonic reactions take up

free energy and have a positive ΔG.

Endergonic reactions proceed

only if free energy is provided

Equilibrium in Reversible Reactions

The change in free energy (ΔG) of a reaction determines its point of chemical equilibrium, at

which the forward and reverse reactions proceed at the same rate.

For exergonic reactions, the equilibrium point lies toward completion (the conversion of all reactants

into products).

@ 0.02M, 25C & pH 7ΔG = -1.7 kcal/mol

ATP Hydrolysis

ATP (adenosine triphosphate) serves as an energy currency

in cells.

Hydrolysis of ATP releases a relatively large amount of free

energy (-12 kcal/mol).

Oxidation of Luciferin in fire flies is powered by ATP

hydrolysis.

ATP Hydrolysis Is Coupled

Biological Catalysts are Enzymes

Change in free energy (ΔG) is indicative of equilibrium point.

The more negative ΔG is, the further the reaction proceeds towards completion.

However ΔG does not tell us any thing about the rate of a reaction (the speed at which it moves towards

equilibrium).

Most Enzymes are proteins (ribozymes are RNA)

Activation Energy

Exergonic although release energy however they are not generally

spontaneous and proceed only after the reactants

are pushed over the energy barrier by small

amount of added energy (Activation Energy Ea

e.g. CH4 + O2

Activation Energy

Activation energy changes reactants into unstable molecules forms called transition-state species.

At normal temperatures only few molecules have enough kinetic

energy for this transformation.

Enzymes lower the energy barrier.

Enzymes are Specific

Reactants (substrates) bind the a particular (active) site of the respective enzyme.

The specificity of this binding is a function of three-dimensional shape, structure and environment of its

active site.

Enzymes Lower the Energy Barrier

Enzymes Lower the Energy Barrier for both forward and reverse reaction.

Equilibrium and ΔG are not changed by enzymes.

50% 600 poly arginine

degradation by carboxypeptidase 7 years vs. half a

second

How do Enzymes Work

How do Enzymes Work

Induced Fit

Upon binding to substrate, some enzymes change shape, facilitating catalysis.

The shape changes (Induced fitting) modify the active site and exposes/ aligns those regions that

perform catalysis

Water is exclusion at the active site results in transfer

of Pi from ATP to glucose preventing APT hydrolysis

to ADP The active site = the catalytic + binding sites

Helpers

Cofactors: Inorganic ions (Cu, Zn & Fe) bound to some enzymes and are essential to their function.

Coenzymes: Carbon containing molecules bind enzymes temporarily (by collision) and are

chemically changed during reaction (like substrate).

Prosthetic groups: non-protein component permanently bound

to enzymes (heme group), without its prosthetic group apoprotein vs. holoprotein.

Cofactors and Coenzymes

Substrate Concentration

Enzyme Regulation

Some factors can activate enzymes and other can deactivate them (inhibit their function).

Irreversible inhibitors permanently inhibit enzymes function generally by modifying the active site

Reversible Inhibitors

Competitive inhibiters are similar to the natural substrate (bind the active site) however they are different enough that no reaction is catalyzed.

When their concentration is lower they come of the enzyme and enzyme can bind the substrate (ADH).

The Citric Acid Cycle

Reversible Inhibitors

Noncompetitive inhibiters bind the enzyme at a a site other than active site and cause a

conformational change that prevents enzyme from binding its substrate or slow the catalysis.

Feed Back/End Product Inhibition

End product can serves as allosteric inhibitor of commitment step enzyme If sufficient amount of end product of a pathway is available in the environment

Allosteric Regulators

Allostery (different shape) change in enzyme shape due to binding of a noncompetitive inhibiter.

These enzymes exist in more than one shape and have

multiple poly peptide chains.

Allosteric regulator (AR) can be positive or negative.

Enzymes catalyzing commitment step have AR

Physical Factors Influence Enzymes

Fever?