7 - Enzymes II The active site

Question 1

Transition state analogues; formation of the enzyme-substrate complex

Answer 1

• First step in catalysis is the formation of the enzyme-substrate complex, ES.
• Enzymes work effectively by reducing the energy (EA) needed to reach the transition state of the substrate.

Question 2

Pauling’s hypothesis

Answer 2

predicts that the transition state binds more strongly to the enzyme than the substrate
• Many examples are now known of molecules that are similar in structure to the transition state– Transition state analogues
• They have very high affinities for enzymes

Question 3

Give 2 examples of transition state analogues

Answer 3

• yeast aldolase

- adenosine deaminase

Question 4

Who proposed the induced fit hypothesis?

Answer 4

Daniel Koschland (1958)

Question 5

What is the active site?

Answer 5

The region of the enzyme that binds the substrate (and any cofactors)

Question 6

Amino acids in the active site come from the same locations in the primary amino acid sequence - true or false

Answer 6

false - they come from different locations in the primary amino acid sequence

Question 7

How do amino acid orientation promote catalysis?

Answer 7

the folding of the polypeptide chain that brings the amino acids into close proximity

Question 8

What type of interactions bond enzyme and substrate?

Answer 8

Bonds formed between enzyme and substrate are mostly ionic bonds between amino acid side chains in the enzyme and the substrate along with the occasional hydrogen bond. NOT COVALENT

Question 9

The active site is usually a 3D pocket or groove in the structure of the enzyme - true or false?

Answer 9

true

Question 10

How does the active site facilitate reactions?

Answer 10

1. Entropy effect
2. Orbital steering
3. Induced fit

Question 11

Entropy effect

Answer 11

Substrates held next to each other or catalytic groups for increased length of time
- e.g. kinase on previous slide – ATP and protein substrate random unless enzyme present – disorder i.e. entropy decreased

Question 12

Orbital steering

Answer 12

Best orientation of substrate relative to catalytic groups.

Question 13

Induced fit

Answer 13

Maximal binding involves changes to conformation of E and S.

• Promotes formation of transition state (reduces activation energy)
• improves specificity

Question 14

How does induced fit improve specificity?

Answer 14

• Open conformation allows substrate binding.
• Closed conformation reconstitutes catalytic site
- Promotes formation of transition state (reduces activation energy)

Question 15

Apoenzyme

Answer 15

• Some enzymes need an additional non-protein component to work properly
• Without these components – inactive ‘apoenzyme’

Question 16

Holoenzyme

Answer 16

enzyme with its cofactor (active)

Question 17

Coenzymes

Answer 17

• non-protein organic
• bind at CATALYTIC SITE
• normally reversibly bound
• a few permanent – prosthetic group e.g. haem in haemoglobin

Question 18

Cofactors

Answer 18

• inorganic – mostly metals (Cu+, Zn2+, Fe2+, Mg2+, K+, Ca2+)
• bind not at catalytic site
• near or in active site though
  Cofactors extend the range of chemical reactions that enzymes can catalyse

Question 19

What type of interactions bond enzyme and substrate?

Answer 19

Bonds formed between enzyme and substrate are mostly ionic bonds between amino acid side chains in the enzyme and the substrate along with the occasional hydrogen bond. NOT COVALENT

Question 20

Cofactor (metal ion) function

Answer 20

• Assist in substrate binding, stabilization (also of transition state) and orientation
• Also stabilize the catalytic site (hold it in the right conformation)
• Can also participate in some reaction mechanisms; general acid-base catalysis and oxidation-reduction reactions

Question 21

3 main types of reaction mechanisms

Answer 21

• Acid-base catalysis
• Covalent catalysis
• Electrostatic catalysis (including metal ion catalysis)

Question 22

General acid/base catalysis

Answer 22

• Involves acidic and basic groups of amino acids in the active sites of the enzyme. Mostly histidine – acid (H+ donor) and base (H+ acceptor)
• Chymotrypsin – aspartate, histidine and serine catalytic triad

Question 23

Covalent catalysis

Answer 23

• A covalent link is formed between substrate and AA in catalytic site of the enzyme e.g. Chymotrypsin (again)
• Amino acids commonly involved (nucleophilic):Serine (-OH), Cysteine (-SH), Histidine (imidazole ring), Lysine (-NH2
• Bond formation contributes to EA

Question 24

Electrostatic catalysis

Answer 24

• Favours formation of transition state by stabilizing its structure – ionic bond formation between enzyme and substrate
• Acidic or basic AAs (lysine, arginine, aspartic acid, glutamic acid) and/or a metal cofactor such as zinc
• e.g. zinc metalloproteinase (partial mechanism shown)
• Cleaves peptide substrate (pink)
• Amino acids shown – glutamate (yellow), histidines (white)
• Zinc co-ordinated by x 3 His

Question 25

ow do enzymes

increase reaction rates?

Answer 25

• Holding substrates together
– Decrease entropy / orbital steering
• Promote formation of transition state
– Induced fit
• Contribute reactive groups
– Amino acid side chains
– Cofactors

Question 26

Michaelis-Menten; the assumptions and limitations

Answer 26

Assumption/limitation 2 – k2 in the simplified equation above assumes that there is no reverse component to this step of the mechanism but:

1. REVERSE CATALYSIS – a number of enzymes can catalyse the conversion of the product back into substrate.
2. PRODUCT INHIBITION

Question 27

The importance of using initial reaction rates; mitigating reverse catalysis and product inhibition

Answer 27

• V0 = initial velocity (initial reaction rate)
• Experiment (right) – measure product formation over time at 4 different
substrate concentrations
• Use initial velocity to plot on MM graph – overcomes issues of reverse
catalysis and product inhibition (product negligible in early stages of