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Zymogen activation by proteolytic cleavage

  • To prevent activation where not wanted (see yesterday's lecture)

Digestion of dietary fat


  • Lipases are water soluble, so they don't easily come into contact with the lipids
    • Can't interact with TAG without micelles
  • Lipase's active site is like a lid - needs colipase to ctivate it and opens the lid
  • Break lipids (TAG) into fatty acids
  • Lipase is inactive without the colipase

Lipase and colipase

Lipase and colipase
  • Colipase is secreted with the pancreatic lipase. It binds to the inactive lipase and helps to bind it to lipid micelles
  • It also causes a conformational change in the lipase, exposing an active site in the region of the enzyme surface in contact with the micelle and "switching on" the enzyme.
  • Bile salts are like detergent, enable the water soluble lipase to contact the lipids (by forming lipid micelles)

Possible mechanisms for the control of enzyme activity

  • Alter the activity of existing enzymes by the reversible binding of (small) molecules
  • Alter the activity of existing enzymes by covalent modification
  • Alter the total amount of an enzyme present

Control of enzyme activity - via reversible binding

  • Competitive inhibition is a common mechanism for drug action

Regulation of trypsin

  • Want trypsin to be actively breaking down proteins in the gut, not in the pancreas
    • The trypsin inhibitor binds to the active site (so it is a competitive inhibitor)
    • It is a substrate, but binds so well that you don't get turnover for a long time

Control of enzyme activity - via reversible binding

  • Competitive inhibitors (substrate analogues) bind at the active site. This is not a common mechanism for controlling the activity of intracellular enzymes, BUT it is a common mechanism for drug action
  • Activation and inhibition of intracellular enzymes with "allosteric" properties is a general mechanism for controlling the metabolism to meet the internal requirements of the cell

Control of intracellular enzymes via allosteric properties

  • Many intracellular enzymes that are important control points in metabolism have allosteric properties
  • Allosteric enzymes invariable have quaternary structure - they have two or more subunits with active sites

Allosteric enzymes - mechanism

Allosteric enzymes mechanism
  • Allosteric enzymes oscillate between an active and an inactive conformation
  • Allosteric activators and inhibitors bind reversibly and lock the enzyme in the active or inactive conformation

Factors affecting enzyme activity

Allosteric enzymes have an "S" shaped curve of reaction velocity vs substrate concentration
  • Enzyme concentration [E]
  • Temperature
  • pH
  • Substrate concentration [S]
  • Cofactor requirements
  • Inhibitors
  • Sigmoid shaped curve - due to the quarternery structure of the allosteric enzyme (compare to the black curve of a non-allosteric enzyme)

Control of glucose breakdown

  • The enzyme activity can be altered over a range of ~20 fold by the allosteric activity
  • Activator: F-2,6-BP, changes enzyme kinetics

Control of enzyme activity - via covalent modification

  • Cleavage of specific peptide bonds, e.g. zymogen activation in protein digestion and blood clotting
  • A chemical reaction with a particular amino acid side chain, e.g. phosphorylation and dephosphorylation

Control of intracellular enzymes via phosphorylation

  • Phosphorylation and dephosphorylation of hydroxyl (-OH) groups on amino acid side chains is a common mechanism for controlling enzyme activity (and the functions of other proteins such as receptors and transporters).
  • Activity (function) can be switched "on" or "off" in different proteins by phosphorylation
  • Receptors (e.g. insulin receptors) span the membrane, often bound receptors switch on other proteins in the cell, by phosphorylating them

Phosphorylation of serines by protein kinase A


  • Phosphorylation results in a conformational change, activating (or inhibiting) the enzyme, and can be reversed by a phosphatase
  • ATP often provides the P group for phosphorylation

Role of protein phosphorylation

  • Phosphorylation/dephosphorylation by protein kinases and phosphatases commonly occurs as part of cellular responses to external signals, such as the binding or hormones or neurotransmitters to receptors
  • It is one of the ways of changing the activity of an enzyme (or the function of other types of proteins) to control metabolism or other cellular processes in response to external signals

Control of triacylglycerol breakdown in adipose tissue


  • 7TM receptor = a GPCR (alpha, beta, gamma), one subunit breaks off when hormone binds, this subunit binds to adenylate cyclase, causes ATP to be metabolised to cAMP. cAMP activates protein kinases, which phosphorylates (and activates) two proteins:
    • Hormone sensitive lipase (in response to glucagon/adrenaline - converts DAG to MAG)
    • Perilipin (activates TAG lipase by phosphorylating it. TAG lipase converts TAG to MAG).

(MAG is then broken down by MAG lipase).

  • Amplification process: just need a small amount of hormone as our signal to activate thousands of catabolic enzymes
  • This is a typical signal transduction pathway

Protein degradation allows amino acids to be reused

  • No means to store amino acids (unlike fat, carb). Need a constant supply for them.
  • The amino acids come from dietary protein and the degradation of cellular proteins

Control of enzyme activity via altering amount of enzyme

  • The control of gene expression (synthesis of new protein)
  • The rate of intracellular enzyme degradation and removal via ubiquitin-tagging and proteasome digestion

Protein degradation

  • Protein turnover occurs constantly
  • Proteins have varying half-lives
    • Crystallin - the life of the organism
    • Ornithine decarboxylase - 11 minutes
  • E.g. cyclin has to be completely degraded before a cell can enter anaphase

Protein degradation regulates biological functions

  • Gene transcription
  • Cell-cycle progression
  • Organ formation
  • Circadian rhythms
  • Inflammatory response
  • Tumor suppression
  • Cholesterol metabolism
  • Antigen processing

Ubiquitin (Ub) marks proteins for degradation

  • Enable protein turnover to be tightly regulated
  • Found in all eukaryotic cells
  • Highly conserved
    • Yeast and human Ub differ by only 3 out of 76 residues
  • Attaches to target proteins with the assistance of three enzymes (E1, E2 and E3)

  • E2 is a reader- it recognises the protein to be degraded and attaches it to the complex for targeting

Ub conjugation


Attachment of Ub to target

  • E1 - Ub-activating enzyme
  • E2 - Ub-conjugating enzyme
  • E3 - Ub-protein ligase
  • Ub first attaches to a cysteine residue of E1 in an ATP-dependent reaction
  • Ub is transferred to a cysteine in E2
  • E3 transfers Ub to a lysine residue on target the protein

Control of enzyme activity via altering amount of enzyme

  • The control of gene expression (synthesis of new protein)
  • The rate of intracellular enzyme degradation and removal via ubiquitin-tagging and proteasome digestion

Ubiquitin molecules form chains

Ubiquitin molecules form chains
  • The attachment of one Ub molecule is only a weak signal for protein degradation
  • Chains of four or more Ub molecules enhance the signal
  • More ubiquitin binding to the ubiquitin stuck to the target, producing stronger signalling for degradation

What determines whether ubiquitin is attached?

  • The N-terminal residue (not always methionine, because there is post-translational modification of proteins)
  • PEST boxes (proline, glutamic acid, serine, threonine)
  • Cyclin destruction boxes (particular amino acids present in cyclin that make it be degraded)
    • Particular features of the protein make it a target for targeting for degradation
  • Juvenile-onset Parkinson's is associated with defective E3 protein
  • HPV (90% of cervical cancer)-encodes a protein that activates an E3, which becomes overactive and attaches ubiquitins a lot more than it should (including attaching it to p53 and DNA repair proteins)

Proteasome degrades the Ub-marked protein

The structure of the proteasome
  • A 26s proteasome complex digests Ub-tagged proteins
  • 20s proteasome - catalytic activity
  • 19s regulatory subunit
  • Two parts:
    • Regulatory portion - recognises the ubiquitin target, unfolds the protein, cuts off the ubiquitin tag and recycles it. Target protein is fed into the inside of the barrel, and goes through multiple rounds of ATP hydrolysis until it becomes 9-12 amino-acid long sequences (recycling or antigen presentation)


26s proteasome function in protein degradation
  • Ub-tagged proteins are unfolded and progressively digested
  • Multiple rounds of ATP hydrolysis occur
  • Ub is spared and can be recycled
  • The beta-subunits of the proteasome split the tagged protein into small peptides and releases ubiquitin for re-used
  • The peptides are then broken down to amino acids by other peptidases