BIOC 212 Lecture Notes - Lecture 10: Reduction Potential, Thiol, Ribose
28 May 2018
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6- Membrane Proteins
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Protein Disulfide Isomerase
• Protein disulfide isomerase (PDI): member of the thioredoxin family
• ER lumen is an oxidizing environment, but spontaneous disulfide bond formation
is inefficient and/or incorrect for folding
o In contrast to cytosol and nucleus, which are reducing environments
o Favors formation of disulfide bonds since it is an oxidation reaction
o Natural disulfide bonding is not efficient enough for normal cellular growth
• Spontaneous disulfide bonding can be incorrect
• Can have multiple cysteines in a protein, not all cysteine connections will
be the right ones
• Even though formation of disulfide bonds is already favored, thioredoxins help
speed up the formation
• Disulfide formation is catalyzed by thioredoxins:
o PDI and ERp57
o Catalyze rearrangement of disulfide bonds until get right combination
o PDI from yeast
o Have 2 reactive Cys residues pointing in to the space, can oxidize substrate
• Cysteine residues have ability to oxidize the substrate
Disulfide Isomerization
• Oxidized PDI catalyzes formation of disulfide bonds in substrate
o Begins itself in the oxidized state
• PDI becomes reduced
• Reduced PDI aids rearrangement of disulfide bonds in substrate during folding
o The first disulfides to form spontaneously may not be correct for the native
state
o i.e. In picture on the right, have 4 cysteines in a protein
• Form disulfide bonds, but are not the right ones --for native folding to
occur, need to form a different set of disulfide
• PDI reacts with the substrate, forming a temporary disulfide bond
between itself and the substrate
• Help switch the arrangement of the disulfide bonds
• When finish the reaction, back to same state of PDI as when started
o Native disulfides are the most stable
• PDI will keep rearranging these disulfides, but once are in the native
state, are in the most stable form so even if try to rearrange the disulfide
it will not do anything
• When partially folded, rearranging the disulfides will help
• PDI therefore helps folding
• In both reactions, PDI forms mixed disulfide bond between intermediate & itself
• PDI with 2 oxidized cysteine residues linked by disulfide bond (S-S)
o Substrate has two cysteines, in the reduced state (sulfhydryl groups)
• During reaction, PDI forms a temporary disulfide bond between itself & substrate
• When finishes, end up with disulfide bond on the substrate
o PDI becomes reduced
• Transferred the oxidizing equivalent from PDI to substrate
PDI Regeneration
• During reaction, PDI becomes reduced after oxidizing the substrate
o So to maintain homeostasis, must find a way to regenerate the PDI
o A chemical cascade regenerates oxidized PDI
• PDI beings in the oxidized state, and becomes reduced when forms S-S
o Mechanism that takes the reduced PD1 and re-oxidize it
o Done by protein Ero1 (ER oxidation)
• PDI is oxidized by Ero1 protein with cofactor FAD
• Ero1 is regenerated by FAD
• FAD is regenerated by O2
• Oxidation potential in ER is maintained, but controlled by enzymes (PDI, Ero1)
• Ero1 starts in the oxidized form, oxidizes PDI and becomes reduced
• Ero1 itself becomes regenerated by co-factor molecule FAD
• Ero1 back to oxidized state, while FAD is reduced
• FAD itself is regenerated by molecular oxygen
• Oxygen diffusing throughout the cell
• End up with hydrogen peroxide (H2O2)
• Cascade of oxidation will help maintain the oxidation of PDI in the ER
FAD
• Flavine Adenine Dinucleotide (FAD)
• Adenine with ribose, phosphate, phosphate, a bunch of stuff, and a flavine
which is redox active
• Analogous to Co-enzyme A (ADP-linker-SH)
• ADP as a handle, an extension, and a reactive group in the end
• Flavine goes back and forth between reduced & oxidized states
• Carrier molecule: ADP-linker-flavine with redox active N sites
• Enzymes that use FAD, bind to the ADP part like a handle, and have the
reactive part sticking out on the outside where it can react with whatever it
needs to
Document Summary
Protein disulfide isomerase: protein disulfide isomerase (pdi): member of the thioredoxin family, er lumen is an oxidizing environment, but spontaneous disulfide bond formation is inefficient and/or incorrect for folding. If the glucose is there, that means the folding is incomplete, and calnexin will keep it into the er so can continue folding. If glucose is absent, means that finished and can move on: non-covalent interactions; always coming on & off very quickly, other enzyme that removes the glucose, eventually all mannose chains will have glucose removed. If non-native, uggt will recognize the polypeptide and put the glucose back on such that calnexin can bind again: keep protein in the er. If in native state, then not recognized by enzyme uggt so glucose not put back on --calnexin will not bind. If have mannose chain with no glucose and are folded, not bound by calnexin anymore, and can proceed to leave the er.
