Biochemistry Online: An Approach Based on Chemical Logic

Biochemistry Online





Learning Goals/Objectives for Chapter 7B:  After class and this reading, students will be able to

  • identify the types of catalysis used in enzyme-catalyzed reactions given a detailed mechanism;
  • interpret kinetic experiments experiments varying substrate, inhibitors, pH, ion strength, and amino acid side chains (through chemical modification or site-specific mutagenesis) to better understand the catalytic mechanisms utilized in enzyme-catalyzed reactions;
  • identify potential rate limiting steps in enzyme catalyzed reaction mechanisms and simplify kinetic equations based on them;
  • generally describe the diversity, the critical active site residues and the biological activities of proteases;
  • describe the structure/function of the proteasome.

B5.  Protease Activation and the Proteasome

Protease are potentially dangerous if their activity is not regulated.  A common method to avoid unwanted protease activity is to activate the enzyme from an inactive precursor called a zymogen.   The precursor is often called a proenyzme.  Limited but regulated proteolysis of the proenzyme by either a different protease or by autoproteolysis leads to activation of the proteolytic activity of the zymogen.  One important example is activation of procaspases to active caspases, which are calcium activated cysteine proteases  that are homodimers.  Activation of caspases initiates programmed cell death.  Cancer cells which are immortal have found ways to inhibit procaspase activation.  Hence a possible cancer therapy could involve drug-induced activation of procaspases.  Wolan et used  high-thoroughput screening to identify compounds that promote the activation of procaspase-3 at physiological conditions and concentrations. A dozen compounds were found to promote such activity, and their ability to activate other similar enzymes in the procaspase family were explored.  Active caspases appear to be in equilibrium between an active state and an inactive one more similar to the inactive zymogen.  Small drug that bind preferentially to the active state would shift the equilibrium from the inactive state to the active state.  Likewise it might bind to the inactive zymogen and promote an "active" conformation of the zymogen leading to the actual activation of the zymogene by autoproteolysis.  Wolan et al discovered that procaspase was able to undergo a conformational change with the addition of a specific small molecule activator (referred to as 1541) which made the “on (active)” conformation more likely, and therefore encouraged self-activation.

Proteases are found in both extracellular (digestive tract, blood, extracellular matrix), membrane, and and intracellular locations.  As mentioned previously, one role of intracellular proteases is to degrade "older" and chemically damaged proteins.   One of the main proteases involved in such intracellular proteolysis is the large protein complex called the proteasome.  It consist of three large structures

Proteins destined for cleavage by the proteasome must first be chemically modified through attachment of multiple copies of the 8,500 MW protein ubiquitin, a highly conserved protein found ubiquitously in eukaryotes.  (We modeled this protein in the first lab using VMD and NAMD.) The carboxyl  group  of the C-terminal Gly residue of ubiquitin forms an amide link to the side chain amine group of Lys  residues in the protein targeted for degradation.  The resulting link is an isopeptide bond since the N terminal of the target protein is not used in the amide bond.  Three different proteins are involved in the ubiqutinylation of the target protein, including E1 (ubiquitin-activating enzyme which requires ATP), E2 (ubiquitin conjugating enzyme) and E3 (ubiquitin-protein lyase).   Once attached, a side chain Lys of ubiquitin can form another isopeptide bond to a C-terminal carboxyl group of another ubiquitin, forming a growing ubiquitin chain on the target protein.  Proteins with 4 or more linked ubiquitins are better substrates for the proteasome.  Proteins with short half-lives (those with certain amino terminal amino acids like arginine or leucine, or enriched in Pro (P), Glu (E), Ser (S), and Thr (T) - (PEST) appear to be better targets for the ubiquitin pathway and subsequent degradation by the proteasome.

Proteasome activity is intimately related to health and disease.  A major role of the proteasome occurs in immune recognition of self and nonself.  The immune system must be able to recognize a virally infected or tumor cell (both self cells expressing foreign or aberrant proteins) as well as foreign cells like bacteria, which can be engulfed by immune cells such as macrophages.  Proteasome involvement  occurs when viral, tumor, or bacterial proteins are degraded to short peptides, which bind intercellular major histocompatability proteins (MHC) proteins and are translocated to the cell membrane.  Peptide/MHC complexes are displayed on the cell surface and are recognized by receptors on immune cells (specifically T cells).  Self cells are not recognized by T cells since the peptides in the peptide:MHC complex are self peptides derived from normal proteins.   The T cell receptor recognizes determinants on both the MHC protein and the presented  peptide. 

animation of protein processing and display of peptides by MHC proteins on cell surface

HHMI animation of the ubiquitin and the proteasome

Nonrecognition of self peptide:MHC complexes prevents the immune system from targeting normal healthy cells.  Autoimmune diseases arise when the T cell receptor recognizes presented self peptides. 

The ubiquitin/proteasome pathways have been linked to disease manifestation in many neurodegenerative diseases like Alzheimers, Huntington's disease (which involves the aberrant folding of the Huntington protein which contains an expanded poly-Glu domain), and Parkinsons.  The degradation pathway is involved in many other normal cellular functions including gene transcription and programmed cell death.

 JmolUpdated Mammalian Proteasome  - (1IRU)   Jmol14 (Java) |  JSMol  (HTML5) 


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 Archived version of full Chapter 7B:  Mechanisms of Enyzme Catalysis

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