Enzymes I and II (The Living Cell)

• Digestion: carbohydrates, fats, proteins

• Blood clotting: fibrin clot catalysed by thrombin

• Defence-immune system-activation of complement

• Movement: muscle actomyosin is an ATPase

• Nerve conduction: membrane pumps for Na+, K+, Ca++

• phenylketonuria-cannot convert Phe to Tyr.

• glycogen storage disease-cannot mobilise glucose

• Tay-Sachs disease-defect in processing a membrane ganglioside, which results in neuronal damage and death

Inhibit cell wall synthesis

Blocks prostaglandin synthesis

(a group of lipids with hormone-like actions that your body makes primarily at sites of tissue damage or infection that influences blood clotting.)

It is a folate analogue which interferes with synthesis of DNA precursors

Increase reaction rate by up to10 billion-fold!

• Show specificity

• Unchanged at end of reaction

• Do not alter reaction equilibrium

• Enzymes speed up reaction by decreasing the free energy of activation of the reaction

•Have active sites that bind substrate(s)

The acceleration of a chemical reaction by a biological catalyst, the enzyme

Enzyme-substrate binding energy

• To bring molecules together in active site

• To constrain substrate movement

• To strain particular bonds in the substrate

• To stabilise positive and negative charges in transition state

• To exclude water from the active site-make reaction go faster

• To provide a reaction pathway of lower energy

• Use cofactors

-Polysaccharide cleavage causes bacterial rupture and death

• The active site is a 3-D cavity or cleft that binds substrate(s)with specificity through electrostatic, hydrophobic, hydrogen bonding and van der Waals interactions.

• X-ray crystallography (usually done at synchrotrons-Diamond, Oxford)

• Kinetic studies of enzyme activity (simpler and quicker than crystallography)

Substrate glucose to Enzyme Hexokinase

Michaelis-Menten Kinetics

Describes the relationship between the rate of an enzyme-catalysed reaction and the concentration of substrate.

The Michaelis-Menten equation is expressed as (picture):

where:

V0 is the initial reaction velocity.

Vmax​ is the maximum reaction velocity.

[S] is the substrate concentration.

Km​ is the Michaelis constant, representing the substrate concentration at which the reaction velocity is half of Vmax​.

k1​:

Represents the rate constant for the formation of the enzyme-substrate complex (ES) from the free enzyme (E) and substrate (S).

E+S→ES

k2​:

Denotes the rate constant for the conversion of the enzyme-substrate complex (ES) into product (P) and free enzyme (E).

ES→E+P

Describes the efficiency of the catalytic step, specifically the breakdown of the enzyme-substrate complex.

kcat​ (Turnover Number):

Represents the rate constant for the conversion of enzyme-substrate complex (ES) into product (P) and free enzyme (E).

ES→E+P

Reflects the maximum catalytic efficiency of the enzyme, indicating how many substrate molecules an enzyme can convert into product per active site per unit time

Max no of substrate molecules handled per activesite per second

A measure of substrate binding affinity-in μM (micromolar) to mM range (millimolar)

Competitive vs non-competitive inhibitors-valuable in drug design work

kcat for most enzymes is ~10 s -1

An Inactive EI complex

Km is increased (it takes more substrate to achieve Vmax/2).

However, Vmax is unaltered, as the effects of the inhibitor can be competed out at high substrate concentrations.

• Control of gene expression-enzyme amount

• Compartmentation: sequences in enzyme polypeptidechain target enzyme to ER, mitochondrion, nucleus etc

• Allosteric regulation: a regulatory molecule (acting at apocket distinct from the active site) changes the enzymeconformation to influence the active site and decrease (or insome cases, increase) enzyme activity. Controls the fluxof material through a metabolic pathway.

• Covalent modification of enzyme. Change enzyme shape and activity-e.g. phosphorylation

• Multisubunit complexes• Regulatory sites and catalytic sites ondifferent subunits• Regulation occurs via conformational changes• Exhibit non-Michaelis-Menten kinetics:V vs S plots are sigmoidal• Involved in feedback inhibition of metabolic pathways

Novobiocin

Methotrexate-an anticancer folate analogue

Catalytic efficiency

-When evolution has sped up the 'chemical' step to the greatest degree

Diffusion

Carbonic anhydrase

Hydrolysis

A charge-relay system by proton withdrawal

Attacks the peptide bond to form an acyl-enzyme

-3

-A rotating spindle

Yes

Kills Mycobacterium tuberculosis by inhibiting its ATP synthase.

An enzyme clamp that unlinks daughter chromosomes

Cross, a DNA segment from one chromosome through a transient ds-DNA break in another (a temporary or short-lived interruption in both strands of the DNA double helix).

Actin:

Forms thin filaments in muscle fibres.

Has binding sites for myosin heads.

Undergoes conformational changes during muscle contraction.

Myosin:

Forms thick filaments in muscle fibres.

Possesses myosin heads that interact with actin.

Utilizes ATP hydrolysis to generate force and movement.

Cross-bridges between actin and myosin contribute to muscle contraction.

Neural Stimulation:

Initiated by nerve impulse, releasing acetylcholine at neuromuscular junction.

Acetylcholine binds to sarcolemma receptors on the muscle cell membrane.

Action Potential Propagation:

Action potential travels along sarcolemma and into transverse tubules (T-tubules).

Calcium Release:

Action potential triggers release of calcium ions from sarcoplasmic reticulum (SR).

Troponin and Tropomyosin Interaction:

Calcium binds to troponin, causing conformational changes.

Tropomyosin shifts, exposing myosin-binding sites on actin.

Cross-Bridge Formation:

Myosin heads bind to exposed actin sites, forming cross-bridges.

Contraction:

ATP hydrolysis and power strokes lead to sliding of actin and myosin filaments, inducing muscle contraction.

Relaxation:

Calcium actively transported back into SR

Calcium Signalling:

Calcium influx through cell membrane channels and intracellular stores.

Calmodulin Activation:

Calcium binds to calmodulin, forming the calcium-calmodulin complex.

MLCK Activation:

Calcium-calmodulin activates Myosin Light Chain Kinase (MLCK).

Myosin Phosphorylation:

MLCK phosphorylates myosin, enabling interaction with actin.

Cross-Bridge Formation:

Myosin-actin cross-bridge formation initiates contraction.

MLCP:

Myosin Light Chain Phosphatase (MLCP) dephosphorylates myosin, promoting relaxation.

Latch State:

Some smooth muscles maintain force with low energy consumption (latch state) due to sustained myosin phosphorylation.

Why Named Serine Proteases:

Contain serine residues in their active sites.

Biological Functions:

Chymotrypsin, trypsin, and elastase play roles in protein digestion.

Chymotrypsin cleaves at hydrophobic residues.

Trypsin cleaves at basic residues.

Elastase cleaves at small, uncharged residues

Definition:

Consists of three amino acids (Serine, Histidine, Aspartate) in the active site.

Function:

Coordinates the catalytic reaction by stabilizing reaction intermediates.

Serine acts as a nucleophile in substrate cleavage.

Set-Up:

Generated during electron transport chain in oxidative phosphorylation.

Protons pumped from matrix to intermembrane space.

Energy Storage:

Potential energy stored in the form of a proton gradient.

Released during ATP synthase-catalysed proton movement back to matrix.

Formation:

Can result from DNA replication or recombination errors.

Examples include catenanes or knot-like structures.

Unlinking by Topoisomerase II:

Topoisomerase II introduces transient double-strand breaks and resolves interlocked structures.

Definition:

Achieves catalysis at the diffusion-limited rate.

Applicability:

Ideal concept; practically unattainable due to limitations such as substrate diffusion.

Thermodynamic Favourability:

Hydrolysis releases energy; however, the rate is slow without enzymes.

Stability in Proteins:

Protein structure and the surrounding environment protect peptide bonds from spontaneous hydrolysis.

Examples and Functions:

Ribosome:

Protein synthesis.

ATP Synthase:

ATP production during oxidative phosphorylation.

DNA Polymerase:

DNA replication.

Helicase:

Unwinds DNA strands during replication and repair.