2 - Enzymes

what is a protein composed of and what bonds hold it together?

composed of 1 or more polypeptide chains, folded into 3D shape, held together by many weak bonds (H bonds, elctrostatic salt bridges, van der waals + hydrophobic interactions)

what are enzymes?

biological catalysts that speed up reactions by lowering the activation energy without altering the final equilibrium between reactants and products.

what is the lock and key theory and is it accurate?

enzyme's active site and a substrate have rigid, complementary geometric shapes that fit together precisely - no it is not accurate

what is the more accurate model?

Induced fit theory

what is the Induced fit theory?

The enzyme’s active site changes shape slightly when the substrate binds, so it fits the substrate more closely.

why is this a better theory?

The lock-and-key model assumes the active site is rigid and already perfectly shaped for the substrate. In reality, enzymes are flexible and change shape when binding, which helps explain how they catalyse reactions more effectively. - forcing it to fit, puts more pressure on the bonds to break.

what is a transition state?

An unstable, high-energy intermediate in a chemical reaction.

what does stabilising the transition state do?

lowers the activation energy needed for a reaction to occur.

why must enzymes be complementary to the transition state?

Reaction without an enzyme: substrate must reach a high-energy transition state before it becomes products (this requires a lot of activation energy and the reaction is slow).

Now if the enzyme was complementary to the substrate, it would bond substrate strongly + stabilise it and it would require even more energy to push it towards the transition state and reaction would not be faster.

Now if the enzyme is complementary to the transition state, enzyme binds substrate and stabilises the transition state (by lowering it’s energy) and decreasing the activation energy + reaction occurs faster.

what is Chymotrypsin?

Endopeptidase = an enzyme that cuts peptide bonds inside a protein chain. endo = inside

what side chains does it prefer?

aromatic side chains - specially

• Phenylalanine (Phe/F)

• Tyrosine (Tyr/Y)

• Tryptophan (Trp/W)

recognises it + cuts after it!

Chymotrypsin is a type of serine protease. whta is that?

a protease that uses a particular serine amino acid in its active site to help break the peptide bond.

Chymotrypsin has 3 important amino acids in it’s active site called the catalytic triad, what are they + what do they do?

Serine + Histidine + Aspartate and they work together to break the peptide bond.

the peptide bond being broken happens in 2 stages, whta are they?

Stage 1: Acylation

Chymotrypsin attacks the peptide bond + one part of the protein is released. The other part temporarily remains attached to the enzyme.

Stage 2: Deacylation

Water is used to break the bond between the remaining part of the protein and the enzyme. The enzyme returns to its original state and is ready to work again.

How specific are enaymes?

They usually only catalyse one type of reaction and work on a few related molecules. Some are so specific that they will only act on one substrate.

what if a natural compound can exist in two stereoisomer forms?

the enzyme will usually act only on one isomer.

Enzyme specificity classification - how many classes are there?

6

what are Oxidoreductases?

They catalyse oxidation/reduction reactions; transfer of H and O atoms or electrons from one substance to another.

what are Transferases?

They catalyse the transfer of functional groups from one substance to another.

what are Hydrolases?

they catalyse the formation of two products from a substrate by hydrolysis (splitting using water).

what are Lyases?

They catalyse non-hydrolytic addition or removal of groups from substrates.

what are Isomerases?

they catalyse the isomerisation changes within a single molecule.

what are Ligases?

They join together two molecules by synthesis of new C-O, C-S, C-N or C-C bonds with simultaneous breakdown of ATP.

what are the effects of temperature on enzymes?

Enzyme structure is stabilised by many weak bonds e.g. H-bonds, electrostatic salt bridges, Van der Waals and hydrophobic interactions. These bonds are easily broken by heating the protein + giving rise to a tangled structure - it is now denatured and no longer has any catalytic activity.

what is optimum ph of an enzyme?

pH where it works fastest.

what are the effects of pH change on enzyme reactions?

pH changes the concentration of H⁺ ions, which can change the charges on amino acid side chains in the enzyme and affects it’s bonding. This may denature the enzyme and reduce it;s catalyitic activity.

what is Enzyme Kinetics?

The study of the rate of an enzyme catalysed reaction - how the rate varies with different substrate concentrations and the effects of inhibitors.

what is the equation for reaction rate?

what is the Michaelis–Menten model?

It describes how the rate of an enzyme reaction changes as substrate concentration increases.

what are the 3 assumptions for this model?

• [S]≫[E] so only a small proportion of the substrate is bound to the enzyme (so cons of substrate pretty much stable throughout reaction)

• Initial velocity is measured: very little product has formed, so the reverse reaction from product to substrate can be ignored.

• Steady-state assumption: the concentration of ES remains constant because its rate of formation equals its rate of breakdown

what is the Michaelis-Menten equation?

when initial velocity = 0.5Vmax, what does that mean for Km and [S]?

they are equal.

what are competitive inhibitors?

• Bind to the active site of the enzyme.

• Compete with the substrate for the active site.

• Often resemble the substrate in shape.

• Their effect can be overcome by increasing substrate concentration because the substrate is more likely to bind to the active site.

what are non-competitive inhibitors?

Non-competitive inhibitors

• Bind to a site other than the active site (an allosteric site).

• Change the enzyme's shape or activity, reducing its ability to catalyse the reaction.

• Can bind to the enzyme whether or not the substrate is bound.

• Their effect cannot be overcome by increasing substrate concentration.

how do Competitive inhibitors affect the graph?

they increase apparent Km + do not alter Vmax

how do non-competitive inhibitors affect the graph?

they decrease Vmax + do not alter Km

what are allosteric enzymes?

enzymes who’s activity is regulated by another molecule binds to them at a site other than the active site (called the allosteric site).

enzyme’s activity can be increased or decreased by activators or inhibitors.

what is feedback inhibition of metabolic pathways?

when the final product of a metabolic pathway binds to an allosteric site on an earlier enzyme, reducing its activity to prevent excess product formation.

what is reversible phosphorylation?

adding or removing a phosphate group.

how does this affect enzyme activity?

it can regulate enzyme activity by activating enzyme or inactivating it.

what do Protein kinases + phosphatases do?

Protein kinases add phosphate groups + protein phosphatases remove them

what are the 4 amino acid residues that can be phosphorylated?

serine (Ser), threonine (Thr), tyrosine (Tyr) and histidine (His).

what are Protein kinases?

enzymes that transfer a phosphate group from ATP onto a protein, a process called phosphorylation, to regulate its activity.

What are protein kinases good targets for?

Drugs

where on the kinase does an ATP molecule attach?

between the kinase's N-terminal and C-terminal domains.

How can you activate a protein kinase?

By phosphorylating it by another kinase.

What is 1 way a cell can regulate enzyme activity?

by changing how much of an enzyme the cell produces.

what is the MAPK cascade an example for?

a kinase cascade, where one kinase activates another kinase downstream.

What does High blood glucose levels stimulate?

insulin production by the pancreas

What does insulin do?

Insulin increases the synthesis/activity of key enzymes involved in glucose metabolism.

Give some examples of those enzymes:

• Glucokinase

• Phosphofructokinase

• Pyruvate kinase