Hydrolytic Enzymes

Question 1

What reaction do hydrolases catalyse?

What are the strong bonds that bind the three main biological polymers?

Answer 1

They catalyse the cleaving of chemical bonds with the addition of water.
Polysaccharides are held together by glycosidic bonds,
Polypeptides are held together by peptide bonds,
Polynucleic acids are held together by photodiester bonds.

Question 2

Hydrolases acting on glycosidic bonds may target what three type of sequences?

Answer 2

Glycogen: repeating glucose monosaccharides (α-1,4 linkage)
Cellulose: repeating glucose monosaccharides (β-1,4 linkage)
Glycosaminoglycans: repeating disaccharides e.g heparin

Question 3

What are the six official groups of enzymes?

Answer 3

EC1-oxidoreductases
EC2-transferases
EC3-hydrolases
EC4-lyases
EC5-isomerases
EC6-ligases

Question 4

Some hydrolases are even more specific than acting on certain polymers. How can they be more specific?

Answer 4

They can be sequence specific, for example restriction enzymes such as EcoR1 or HindIII only act on specific ester bond sequences.

Question 5

An acid anhydride is what?

Answer 5

A molecule with two acyl groups attached to the same oxygen.

Question 6

Name a enzyme that acts on phosphorus containing anhydrides.

Answer 6

ATPase.

Question 7

Give an example of a glycosidase? Explain how it carries out its hydrolytic function.

Answer 7

Lysozyme. It carries out its function by hydrolysing the β(1,4) glycosidic linkages between (N-acetylglucosamine) NAG and (N-acetyl muramic acid) NAM peptidoglycan in bacterial cell walls.

Question 8

How many families of true proteases are there?

Answer 8

There are five families.

Question 9

What are the biological roles of peptide bond hydrolysis?

Answer 9

Digestion of dietary protein,
Activation of proteins, such as proenzymes or zymogens,
Activation of other active proteins such as growth factors or hormones,
Removal of a signal peptide for protein secretion,
Retrovirus polyproteins.

Question 10

HIV cleaves what three polypeptides to release what?

Answer 10

It cleaves the GAG, POL and ENV viral polypeptides to release structural proteins, reverse transcriptase, RNAase and integrase.

Question 11

What are chymotrypsin and trypsin involved in?
What is thrombin involved in?
What do these three enzymes have in common?

Answer 11

Chymotrypsin and trypsin are involved in digestion.
Thrombin is involved in blood clotting.
They all have closely related 3D structures.

Question 12

What does chymotrypsin cleave?
What does trypsin cleave?
What is the P1 residue and what does it bind to?

Answer 12

Chymotrypsin cleaves c-terminally of aromatic residues, such as tryptophan, tyrosine and phenylalanine.
Trypsin cleaves c-terminally of basic residues, such as lysine and Arginine.
The residue that the enzyme targets is known as the P1 residue. This residue binds to the S1 binding site.

Question 13

The active site of all serine proteases contains what, which is made up of what?

Answer 13

Contains a catalytic triad which is made up of a serine residue, a histidine residue and an aspartate residue.

Question 14

How to the residues in the catalytic triad work?

Answer 14

Both histidine and aspartate act together to increase the nuceleophilicity of the serine residue. A proton from the serine hydroxyl is transferred to the imidazole ring of the histidine residue, histidine therefore acting as a base. Aspartate stabilises the positive charge that forms on the histidine residue.

Question 15

During the serine protease mechanism an intermediate is formed, what is this intermediate and what stabilises it the negative charge of one of the oxygen atoms?

Answer 15

A tetrahedral intermediate is formed and it is stabilised by an oxyanion hole, formed by two backbone amide groups of the enzyme.

Question 16

After the acylation step what is released from the catalytic triad and what remains. What allows the deacylation step to occur?

Answer 16

The amine group is released while the carboxylic acid group remains bound to the enzyme. Water is necessary for the deacylation step.

Question 17

What are the two steps of serine protease catalysis?

Answer 17

Acylation and deacylation.

Question 18

Why does the activity of proteolytic need to be controlled? How can the process be controlled?

Answer 18

New peptide bonds cannot be formed so the activity needs to be controlled. This can be done by the protease enzymes being produced as zymogens (inactive precursors)

Question 19

Zymogens are activated by what?
Name two example of zymogens tranformations?
What can these protease activations lead to?

Answer 19

Limited proteolysis- the cleavage of a single peptide bond.
Chymotrypsinogen -> chymotrypsin
Trypsinogen -> trypsin
They can lead to protease cascades of activation.

Question 20

How is haemostasis useful and why is a lack or excess of blood coagulation not good?

Answer 20

Haemostasis is useful because it maintains blood fluidity while preventing blood loss, through clotting.
A lack of blood coagulation can result in haemorrhage and haemophilia. An excess of blood coagulation is not good and may result in thrombosis, heart attack or stroke.

Question 21

What initiates blood coagulation?
How does it prevent blood loss, outline briefly on a protein level?
What enzyme is responsible for blood clotting?

Answer 21

Damage to the vascular system initiates blood coagulation.
The large soluble protein fibrinogen is converted to fibrin which forms an insoluble polymeric meshwork.
The enzyme thrombin is responsible for this protein conversion.

Question 22

Describe the structure of fibrinogen. Removal of what from where results in the molecules conversion to fibrin fibres?

Answer 22

Fibrinogen is made up of 6 polypeptide chains, 2 x α, 2 x β, 2 x γ chains.
The removal of amino terminal peptides from the α and β subunits results in the formation of higher order complexes and the eventual cross-linking that leads to the construction of fibrin fibres.

Question 23

What is coagulation regulated by?
The post-translational modification of coagulation proteases is supervised by what?
Without this molecule the proteins would be unable to bind to what?

Answer 23

Coagulation is regulated by cofactors.
The post-translational modification of coagulation proteases is supervised by vitamin K.
Without this molecule the proteins would be unable to bind to calcium ions, seriously impairing coagulation.

Question 24

Name three cofactors coagulation proteases depend upon.

Coagulation proteases depending on these cofactors are known as what?

Answer 24

Non-enzymatic protein cofactors,
Phopholipids,
Calcium ions,
They are known as vitamin K dependent coagulation factors.

Question 25

Explain why regulatory cascades are useful.

In the case of thrombin what does the cascade do?

Answer 25

Regulatory cascades allow for a very sensitive response to a small molecular signal, with amplification magnifying this response.
The cascade results in the sequential activation of zymogens, each one catalysing the next conversion.

Question 26

Give an outline of the enzymes, in order, of the thrombin regulatory cascade.

Answer 26

Factor XII
Factor XI
Factor IX
Factor VIII
Factor X
Prothrombin activator 
Thrombin 
Fibrinogen -> fibrin

Question 27

Which of the enzymes in the thrombin regulatory cascade are vitamin K dependent coagulation factors?
What do there enzymes contain?

Answer 27

Factor VII, factor IX, prothrombin and factor X are all vitamin K dependent coagulation factors. These factors contain the gamma-carboxyl-glutamate residues.

Question 28

The gamma-carboxyl-glutamate residues are formed via what and what enzyme catalyses this?
What is a cofactor for this enzyme?

Answer 28

It is formed through post translational modification of glutamate residues, this being catalysed by gamma-carboxylase.
Vitamin K is a cofactor for this enzyme.

Question 29

The gamma-carboxy-glutamate residues are strong what in regards to Ca+ ions.
What do they allow coagulation proteases to interact with?

Answer 29

They are strong chelators of Ca2+ ions.
They allow coagulation factors to interact with their cofactors:
Non-enzymatic protein cofactors
Phospholipids

Question 30

What is the final reaction of the thrombin regulatory cascade? Give details of the reactants, the enzyme required and the cofactors required.

Answer 30

It is the reaction in which prothrombin is converted to thrombin and is made of many parts: factor X, Ca2+ (binds to gamma-carboxyglutamate), factor V (protein cofactor) and phospholipid (cofactor provided by cell surface).

Question 31

If the prothrombinase complex is complete how quickly can the conversion of prothrombin to thrombin occur compared to how quickly it can occur with just factor X present?

Answer 31

If the complete complex is present the reaction can be completed in a second. If only factor X is present the reaction takes four days.

Question 32

Why is a vitamin K deficiency unusual and how can it be caused?
What are the symptoms?

Answer 32

It is unusual because vitamin K is found in many leafy vegetables and cereal crops, and bacteria in the GI tract can synthesise it. A deficiency can be caused through improper absorption of the vitamin through the GI tract and long term use of antibiotics.
Symptoms include: bleeding, bruising, oseteoperosis and fractures.

Question 33

How does warfarin act as an anticoagulant?

Answer 33

After carboxylation of glutamate, aided by vitamin K, the oxidised vitamin K is recycled, being first reduced by vitamin K epoxide reductase. Warfarin prevents carboxylation by inhibiting vitamin K epoxide reductase.