Enzymes and co-enzymes Flashcards
Reaction energetics, co-enzymes, enzyme mechanisms.
What are enzymes?
Biological catalysts - carry out all reactions taking place in the cell.
Many inherited diseases are caused by mutations in…
key enzymes.
Measurements of enzymes in blood can be used to…
diagnose disease.
How are enzymes used commercially (3)?
In food products, detergents and drug synthesis.
Enzymes can be split into 6 main groups.
What chemical reactions do oxidoreductases carry out?
Give some example enzymes.
Oxidation-reduction reactions in which oxygen and hydrogen are gained/lost.
Cytochrome oxidase and lactate dehydrogenase.
What chemical reactions do transferases carry out?
Give some example enzymes.
Transfer of functional groups such as an amino group, acetyl group or phosphate group.
Acetate kinase and alanine deaminase.
What chemical reactions do hydrolases carry out?
Give some example enzymes.
Hydrolysis (addition of water).
Lipase, sucrase and beta-galactosidase.
What chemical reactions do lyases carry out?
Give some example enzymes.
Removal of groups of atoms without hydrolysis.
Oxalate decarboxylase and isocitrate lyase.
What chemical reactions do isomerases carry out?
Give some example enzymes.
Rearrangement of atoms within a molecule.
Glucose-phosphate isomerase and alanine racemase.
What chemical reactions do ligases carry out?
Give some example enzymes.
Joining of 2 molecules using energy usually derived from the breakdown of ATP.
Acetyl-CoA synthetase and DNA ligase.
What is the transition state?
A transient (lasting only for a short time) high-energy species in the conversion of substrate to product.
Why is the transition state unstable?
One bond is in the process of being made and the other cleaved.
What does catalysis do to the transition state?
Stabilise it by decreasing energy.
Catalysts lower the activation energy for a reaction but have no effect on…
the Gibb’s free energy change.
Catalysts cannot make a thermodynamically unfavourable reaction become…
favourable.
Catalysts do not change the position of equilibrium but they do…
speed up how quickly the reaction takes to reach equilibrium.
Describe the role of an enzyme as a catalyst in a reaction.
- Enzyme provides specific environment (active site) for substrate where reaction is more favourable.
- Enzyme acts by lowering the activation energy (the energy needed to carry out a reaction).
- Enzyme accelerates conversion of substrate to product and is not used up in the process.
Why is there a peak of energy between E+S and ES?
(look at summary sheet if confused)
Active site has waters in/ the substrate is surrounded by waters and these need to be stripped away.
Why is there another peak between EP and E+P?
Energy is put in to break bonds (usually non-covalent) and release product.
How could the enzyme tightly binding to either the substrate/product limit catalysis?
Enzyme binds to substrate tightly meaning transition state is more stable but also more difficult to release the product.
The active site is designed to complement the reaction…
transition state.
Which forces stabilise the transition state?
The same forces that stabilised protein folding.
e.g. hydrogen bonds, hydrophobic, ionic and van der Waals forces.
In some enzyme mechanisms, the reaction involves the formation of…
a transient covalent bond.
What is binding energy?
The energy derived/obtained from complex formation.
What are co-enzymes?
Give some examples.
Non-protein organic molecules required for catalysis.
e.g. biotin, NAD+ and FAD.
What are co-factors?
Inorganic substances that are required for catalysis.
e.g. metal ions (Fe2+, Zn2+, Cu2+, Mg2+ etc.)
What is a holoenzyme?
Combination of protein and co-enzyme/co-factor.
What is a apoenzyme?
The protein alone, without the co-enzyme/co-factor.
What is the benefit of a co-enzyme/co-factor to an enzyme?
They allow enzymes to access chemistries that amino acid side chains can’t.
What are these examples of?
Co-enzyme A, ATP, S-adenosyl methionine (SAM), pyridoxal phosphate (PLP), thiamine diphosphate (TTP), lipoate, glutathione (GSH), biotin, nicotinamide (NAD+) or phosphorylated nicotinamide (NADP+).
Common co-enzymes.
Formally co-enzymes have a catalytic role and aren’t substrates but the term is used quite loosely so can include…
ATP and NAD+ (which are regenerated in other reactions after being temporarily modified).
For each co-enzyme state the reaction mediated.
Next card.
biotin
carboxylation
cobalamin (B12) co-enzymes
alkylation
co-enzyme A
acyl transfer
flavin co-enzymes
oxidation-reduction
lipoic acid
acyl transfer
nicotinamide coenzymes
oxidation-reduction
pyridoxal co-enzymes
amino group transfer
tetrahydrofolate
one carbon group transfer
thiamine pyrophosphate
aldehyde transfer
What are the following due to:
- megaloblastic anaemia
- beriberi
- pellagra
Deficiencies in co-enzymes:
- folic acid deficiency (used to make
tetrahydrofolate) or B12 deficiency - thiamine (B1) deficiency
- nicotinamide (B3) deficiency
What happens in general acid-base catalysis enzyme mechanisms?
Enzymes stabilise unstable charged transition states (decreasing free energy) by transferring protons to and from them.
For charged transition states, what acts as weak proton donors/acceptors?
Amino acid side chains e.g. Glu, Asp, Lys, Arg, His, Cys, Tyr.
How does His speed up the reaction in the RNAse mechanism?
By stabilising charged intermediates.
Why is the RNAse mechanism necessary?
For the breakdown of RNA in the cell e.g. after mRNA has been translated.
Depending on the stage of the catalysis cycle, two histidines act as…
acids and bases.
Describe the RNAse mechanism.
(If confused, look at printout on summary sheet or watch recap).
- His 12 acts as a general base and takes a proton from RNA 2’ OH to make a nucleophile which attacks the phosphoryl group.
- His 119 acts as a general acid to donate the 5’ hydroxyl proton to the oxygen on phosphate to promote bond scission.
- One product is released and the other remains in the active site.
- His 119 acts as a base by taking proton off water and encouraging the oxygen to attack the phosphorous.
- Electrons from the bond are used to reprotonate the 2’ hydroxyl.
- His 12 acts as a general acid to donate its proton to the 2’ hydroxyl.
What do the numbers mean after His?
Position of residue in polypeptide chain.
Give 3 things the interaction between the enzyme-bound metal and substrate can do.
- orient a substrate for reaction
- stabilise charged reaction states
- mediate oxidation-reduction reactions
What do a third of all enzymes require for catalytic activity?
A metal.
Covalent catalysis involves the formation of…between the enzyme and substrate.
A transient covalent bond.
What does carbonic anhydrase require for catalytic activity?
(look at summary sheet mechanism)
CO2 + H2O ⇌ HCO3^- + H+
Zn^2+ forms a bond with an OH- ion in the active site.
The covalent complex undergoes a reaction to regenerate…
the free enzyme.
In comparison with the uncatalysed reaction, the formation of the covalent bond offers an alternative route to the same products that has a…
lower activation energy.
What are serine proteases?
A large family of enzymes that include digestive enzymes trypsin, chymotrypsin and (blood clotting enzyme) thrombin.
These enzymes are endoproteases that hydrolyse the peptide bond.
They have a catalytic triad (aspartate, histidine and serine) at their active sites.
How do serine proteases hydrolyse proteins?
Using a mixture of acid-base catalysis and covalent catalysis.
What does the serine residue act as?
A nucleophile and is made more nucleophilic by the histidine and aspartic acid.
Give the 6 steps to the mechanism.
- ES complex (Michaelis complex) forms:
- histidine deprotonates serine to make O
more inclined to attack C.
- electrons from double bond transfer to O. - First transition state = tetrahedral
intermediate:
- CN bond cleaved because electrons from
O- reinstate double bond.
- Electrons from cleaved bond form bond
from N to H. - Acyl enzyme intermediate is formed.
- Acyl enzyme water complex:
- histidine works as a base to accept proton
from water.
- water persuaded to attack carbon.
- electrons form single bond by going onto
oxygen. - Second transition state - tetrahedral
intermediate:
- histidine works as an acid to protonate
serine.
- electrons from O- reinstate double bond
and C-O bond is cleaved when electrons
go to proton. - Free enzyme = active.
Each enzyme has a different…
active site.
What does trypsin cleave?
Arg and Lys residues.
What is in trypsin’s binding pocket?
An Asp residue.
What does chymotrypsin cleave?
Aromatic residues.
What is chymotrypsin’s binding pocket?
Serine.
What does elastase cleave?
Small hydrophobic residues e.g. glycine, alanine and valine.
