Please enable JavaScript.

Coggle requires JavaScript to display documents.

Binding - Coggle Diagram

- - - - Emil Fischer, 1894 (no info on proteins), in late 1940s Linus Pauling proposed enzymes facilitate catalysis by binding more tightly to transition state than to substrate and thus lower activation energy
      - the active site is complementary to teh substrate
      - forms an E-S complex
      - but if it was a perfect fit then there is no driving force
    - - Dan Koshland, 1957 (more info on proteins but not atomically)
      - the active site changes on binding the substrate to give an exact fit
      - the protein is flexible and dynamic
  - - - if they are also identical a hyperbola forms (saturation against log(B))
      - if they are not identical then the graph depends on the difference (a large difference produces a double bump, as the difference decreases the nump is less obvious)
      - free energy
        
        when one binds it may cause a larger free enrgy loss compared to if the other bound first
        
        but when both are bound the free energy loss is equal regardless of which binds first
    - - the ligands so that the binding of the first ligand affect future binding
      - Allostery
        
        when the protein has a binding sites that are not in direct contact but the binding on the first will still affect the binding of the next
        
        example
        
        bicyclic crown ether- has 2 benzene rings attached together by a biphenyl group which are also attached to a crown ether ring each
        
        the benzene ring can rotate around themselves and the crown ether rings resist this
        
        it binds 2 mercury compounds in each crown ether ring that show positive cooperactivity
        
        when mercury is added it communicates with oxygens in crown ether to prevent flexing and the biphenyl is now more rigid making second binding easier
        
        the ligand is called an allosteric effector protein of the protein and the protein is called an allosteric protein
      - free energy
        
        first binding of ligand gives same free energy loss as if they were not interacting
        
        but the second gives more negative free energy and enhances binding of second when first is bound (positive cooperativity)
        
        it doesn't matter which ligand binds first
      - without cooperativity the equation is bound to the michalus menson equation
- - - - Lungs have a higher pH and tighter binding than tissues with lower pH (so oxygen affinity falls)
      - the charge-charge interactions, or salt bridge, in deoxyhaemoglobin inhibits oxygen binding
      - at high pH His side chain is not protonated so the salt bridge is not present and oxygen binding is allowed because t-form is not favoured
      - at low pH His side chain becomes protonated and the salt bridge forms which favours the t-form and so oxygen binding
    - - many have bellshaped curve with a maximum pH optimum
      - papain
        
        below 4.2- His159 is positive and Cys25 is uncharged
        
        above 8.2 His159 is uncharged and Cys25 is negative
        
        optimum pH- His159 is positive and Cys25 is negative
  - - - graph for enzyme activity against 1/Temperature
      - it has a sharp increase to peak then a slower decline then at a point of change a sharp decrease
      - the point of change is a lower temperature than optimum and is a change in the rate-limiting step
  - - - example- 2,3, Bisphosphoglycerate which binds to middle of 4 haemoglobin subunit and makes oxygen binding more cooperative
    - - Reversible
        
        These are bound by non-covalent interactions and can dissociate from enzyme to restore activity
        
        They usually resemble the substrate, coenzyme or another metabolic intermediate
        
        They have an inhibition constant when an enzyme-inhibitor complex is formed
        
        Competitive
        
        Binds at the active site and prevents substrate binding
        
        can be countered by increasing substrate concentration
        
        Km increases and Vmax is unchanged
        
        Methotrexate
        
        it is a drug to inhibit nucleotide biosynthesis
        
        it inhibits dihydrofolate reductase which is involved in nucleotide bas synthesis
        
        Non competitive
        
        Binds to enzyme not at the active site
        
        binds equally to free enzyme and enzyme-substrate complex
        
        cannot be countered by increasing substrate concentration
        
        Km and Vmax is unchanged
        
        Uncompetitive
        
        More complex form of inhibition
        
        can bind to ES complex
        
        Km is unchanged and Vmax is reduced
      - Irreversible
        
        enzymes undergo a chemical reaction with the enzyme forming a covalent bond between inhibitor and enzyme
        
        generally, once inactivated it cannot regain activity
  - - - First step is condensation of aspartate and carbamoyl phosphate to give N-carboylasparate catalysed by aspartate transcarbamoylase and is inhibited by CTP (the end product)
      - CTP binding stabilises the T-form of the enzyme so decreases affinity for aspartate and carbamoyl phosphate
      - CTP binds at a site on the enzyme separate from the active site
  - - - acidic interior and is filled with host of hydrolytic enzymes
      - this degradation is particularly targeted to aged or defectives organelles in autophagy, but account for 90% of degradation in mammalian cells
    - - the protein is first marked to target it for proteasomal degradation by ubiquitin
      - the proteasome binds to the targeted protein via the Ub receptors and unfolds the protein using hexameric ATPases and deubiquitinases (these removes ubiquitins) as it is transferred into an internal chamber
      - protein cutting subunits in the chamber degrade the target proteins into small peptides (2-24 residues long) which are then released into cytosol
      - They consist of roughly 60 subunits, have a cylindirical catalytic core called 20S proteasome and bound to both ends or 1 or 2 19S RP complexes (when combine either 26S or 30S
      - the core comprises 2 inner rings of 7 Beta subunits, each ring contains 7 alpha subunits to limit substrate access via core gate. The 3 active sites in each B subunit ring can cleave peptide bonds at hydrophobic, acidic or basic residues
      - the core gate is narrow enough to only let unfolded proteins in, its opening is controlled by ATPase in 19S RP (they aloso control unfolding and translocating them into inner chamber)
      - (inhibition of proteosomes can reveal their function, see textbook)
      - Ubiquitin
        
        proteins are marked by covalently attaching ubiquitin (linear chain of 76 residue copies polypeptide)
        
        Activation of ubiquitin-activating enzyme by addition of ubiquitin using ATP
        
        transfer of this ubiquitin to a ubiquitin-conjugating enzyme's cysteine
        
        ubiquitin protein ligase forms a covalent, isopeptide bond between carboxyl group of Gly-76 of ubiquitin on enzyme and amino group of lys residue on target protein; this occurs multiple times as it adds ubiquitin to lys-48 of previous ubiquitin
  - - - catalysed by protein kinases and removal of phosphates catalysed by phosphatases
      - phosphorylation can influence protein location (e.g. attracting to plasma membrane), its structure, its ability to bind to other molecules (e.g. DNA) or its stability
      - Protein Kinase A
        
        like all other protein kinases, it has a kinase domain (around 250 residus long) that catalyses teh phosphate transfer reaction
        
        A region in the C lobe is called the activation loop and it participates in regulation of activity
        
        Its active site is located around the cleft between the N- and C-lobes, and it contains substrate binding sites and properly orientated ATP on the ledge of the adjacent cleft
        
        When binds, it positions hydroxyl group of phospho-accepting Ser, Thr, or Tyr side chain
        
        binding of substrate place y-phosphate of ATP in close proximity to hydroxyl group and induces a conformational change (induced fit) in N-lobe forming closed conformation
        
        3 negatively charged, acidic side chains in binding site directly bind to Arg side chains in substrate motif and a hydrophobic pocket in PKA recognises hydrophobic side chain in motif adjacent to acceptor Ser or Thr, so only proteins with this motif is phosphorylated
        
        There are also the G- and A-loop, bith involved in binding of ATP-2Mg2+ or the protein enhances binding of other substrate (cooperative and induced fit) and once bound catalytic site transfers y-phosphate to hydroxyl of acceptor at rates 10^10 times faster
        
        Activation
        
        G loop (GXGXXGXV) residues interact with B and y phosphates of ATP and one of the A-loop residues triggers activation
        
        The residues in both loops must be properly orientated in the protein if substrates are to bind adn transfer phosphate to Thr-197 in A loop
        
        this phosphorylation induces conformational change generating active structure
    - - can involve addition of single ubiquitin or multiple
      - All 7 Lys residues (6, 11, 27, 33, 48 and 63), adn N terminal can participate in binding of C-terminal Gly-76 to form the chain
      - multiple form of ubiquitination result in wide variety of recognition surfaces that participate in binding
      - Lys-48 polyubiquitination leads to proteasomal degradtion, lys-63 is used in cellular identification and signalling, lys-11 regulates cell division, lys-33 suppresses T-lymphocyte receptors
    - - Many hormones are synthesised as long precursors and prior to secretion they must be hydrolysed to fold properly
      - sometimes a single long prohormone is cleaved into several distinct active hormones and sometimes are synthesised as zygomenns (inactive precursor enzymes
      - cleavage of peptide bond near N terminus of trypsinogen (zygomen of trypsin) by enzyme generates new N terminal residue (Ile-16) whose amino group forms ionic bonf with carboxylic acid side chain of internal aspartic acid
      - this binding changes substrate binding site which activates enzymes, the active trypsin can then activate trypsinogen, chymotrypsinogen and other zygomens
      - Process of protein self splicing is an autocatalytic process that proceeds by itself in which the excised peptide appears to remove itself from the protein similar to RNA
- - - - binding oxygen causes the haem ring structure to flatten lowering the Kd value fo radditional binding at other subunits
      - this shifts the coordinated side chain of HisF8 and pushes ValFG5 to one side
      - this has a knock on effect on conformation of all 4 subunits
      - Han Frauenfelder called it "protein quake"
- - - - There are 2 binding interactions: substrate and enzyme form H bonds that are similar to B sheet; and a key side chain of substrate that determines which peptide is to be cleaved (and it extends into side-chain-specificity-binding pocket)
      - Trypsin has a marked preference for hydrolysing at carboxyl side of an amino acid with long, positive side chain because side chain is stabilised by negative Asp-189
      - SLight differences of binding pockets explain different substrate specificities: chymotrypsin prefers large aromatic groups since uncharged Ser-189 allows large, uncharged hydrophobuc side chains; elastase prefers small side chain since glycines in side of pocket are replaced with valines (Val-216, Val-190)
      - In all 3 enzymes, Ser-195, His-57, and Asp-102 form catalytic triad that participates in 2 step hydrolysis reaction. Asp-102 and His-57 support attack of hydrocyl oxygen of Ser-195 on carbonyl carbon which forms an unstable transition state with 4 groups attached to carbon, breaking the C-N peptide bond then releases one part of the substratewhile other parts remain attached via ester bond to Ser's oxygen forming stable acyl enzyme intermediate
      - The subsequent replacement of this oxygen with one from H2O leads to release of X-COOH
      - The transition states are stabilised by H bonding with oxyanion hole in enzyme's backbone
      - pH
        
        imidazole of His-57in serine proteases (pKa roughly 6.8) can help Ser-195 attack substrate only if not protonated
        
        so protease activity is low when pH<6.8 at which imidazole is protonated
        
        pH activity curves show an inflection near pH 6.8
        
        activity drops at higher pH values because proper dolding is disrupted when amino group at protein's amino terminus is deprotonated
    - - they are nonpolypeptide small molecule or ion that is bound in the acitive site and plays role in mechanism
      - some can be chemically modified during the reaction and need to be replaced or regenerated (like NAD+, FAD, and haem groups