12 - Enzymes VII: Post-translational modification of enzymes

Question 1

Post-translational modification

Answer 1

covalent attachment of other molecules to the protein after synthesis

Question 2

How do PMTs regulate protein activity

Answer 2

– Phosphorylation
– Acetylation
– Methylation
Reversible modifications allow for continuous regulation

Question 3

Protein phosphorylation

Answer 3

• -OH groups of Ser, Thr and Tyr
– Serine/threonine kinases
– Tyrosine kinases
• Many different protein kinases - 550 in humans

Question 4

Effects of phosphorylation

Answer 4

1. Conformational change

2. Interactions with other proteins

Question 5

1. Conformational change

Answer 5

• Addition of a phosphate group adds 2 negative charges to the protein.
• Alters electrostatic interactions:
– Within the protein - conformational change
– With substrates and regulatory ligands

Question 6

Glycogen synthase

Answer 6

• N- and C-terminal phosphorylation of glycogen synthase- increases negative charge
• Structure of human GS not determined, but modelling suggest change in charge induced conformational change

Question 7

2. Interactions with other proteins

Answer 7

• Regulation of some enzymes by phosphorylation requires the presence of additional factors.
• Phosphorylation alone does not affect enzyme activity, but it promotes the binding of other, regulatory proteins.
• Examples
– SH2 domains found in many different proteins bind phosphotyrosine.
– 14-3-3 proteins bind phosphoserine.

Question 8

Phosphorylation-dependent regulation of plant nitrate reductase by 14-3-3 protein

Answer 8

• Nitrate reductase activity produces toxic nitrite that can only be utilised in the light
• NR kinase is activated in the dark and phosphorylates NR
• Phospho-NR binds 14-3-3, causing enzyme inhibition

Question 9

Proteolytic activation

Answer 9

• Many enzymes (mainly proteases) are converted from inactive precursors into active enzymes.
• This is a permanent activation mechanism.
• Inactive precursors: Zymogens or Proenzymes
• Activated by specific cleavage of one or more peptide bonds.

Question 10

Chymotrypsinogen

Answer 10

• Secreted by the pancreas as a zymogen- common for digestive enzymes
• Trypsin activates many

Question 11

Clots are formed from fibrin

Answer 11

• Fibrin produced from fibrinogen by action of thrombin.

* Thrombin removes A + B fibrinopeptides.

Question 12

Chymotrypsinogen activation

Answer 12

• Only minor changes in tertiary structure
• Disulphide bonds help maintain tertiary structure
• Cleavage site remote from catalytic triad
• Hydrophobic & oxyanion cavities form

Question 13

Blood clotting

Answer 13

• Blood clots are the product of a cascade of zymogen activations.
• Cascades allow amplification of initial weak signals into rapid and large response.
• In the blood clotting response, two cascades feed into the final clotting response:
– Intrinsic response to exposed surfaces of damaged blood vessels,
– Extrinsic response to factors released from damaged tissues.