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Proteins (Bond formation (Affected by (pH: affect intermolecular bonds…

- - - - covalent catalysis: accelerate reaction by catalysing formation of transient covalent bond between enzyme and substrate (active amino acid carries nucleophile that binds to electrophilic site on substrate eg chymotrypsin (serine, histidine and aspartate) acting as triad to facilitate formation of tetrahedral intermediate for reaction.
      - metal ion catalysis: acts as co-factors for binding/orientation of substrates, mediate redox reactions or to stabilise/shield negative charges eg Zn2+ in ACE acts as central metal ion bonded to 2 histidine, 1 glutamate and 1 water ligand and aids to re-orientate products for reaction and activates water molecule to be a stronger nucleophile to attack the carbonyl bond, facilitating peptide hydrolysis.
      - Acid-base catalysis: amino acids around active site donate or accept protons to generate stronger nucleophile/electrophile to facilitate bond formation eg RNase A mechanism (cleaving of RNA) where histidine 12 and 119 work in synergy as acid-base pair to promote nucleophilic attack and bond cleavage. Aspartyl protease (aspartate group acting as acid/base) aids in maturation of viral proteins and are targets of inhibition eg saquinavir
        Properties of amino acids depend on the types of R groups in proximity and weak acids/bases can play a acid-base role
    - - Km value affected by
        
        Presence of competitive inhibitors: higher Km value (more substrate conc to reach same 1/2Vmax value without inhibition) i.e. Vmax (observed) = Vmax x substrate conc/[Km (1 + inhibitor conc/Km in inhibitor) + substrate conc]
        
        Km (observed) = Km/ (1 + inhibitor conc/Kinhibition)
      - Vmax value affected by
        
        Enzyme conc (increase availability of active sites to cope with saturation). Kcat (turnover number) = Vmax/enzyme conc and V = Kcat/Km x enzyme conc x substrate conc
        *Kcat/Km: specificity constant/rate of reaction of active site to form products
        
        Presence of non-competitive inhibitor: Vmax lowered i.e. Vmax (observed) = Vmax / (1 + inhibitor conc/Kinhibition)
        
        Vmax reduced in presence of uncompetitive inhibitor. Vmax (observed) = Vmax / (1 + inhibitor conc/Kinhibition)
    - - Induction
        
        Transcriptional activation
        
        Increase enzyme conc by increasing transcription and translation of genes coding for enzyme eg binding to transcription factors to allow easy binding to promoter region and stimulate transcription process
        
        Allosteric activation
        
        Binding of drug to allosteric site to modulate conformational change of enzyme i.e. exposing active sites to substrate binding, increase ES complex formation eg glucokinase activation to increase blood glucose to glycogen (storage)
        
        Inhibiting drug metabolism process
        
        Metabolites bind irreversibly to enzymes that metabolise drugs (Eg Cytochrome P450 enzymes) by PXR and bergamottin (found in grapefruit), increasing bioavailability of drugs eg amiodarone (anti-hypertensive), cyclosporin (immunosuppressant) and statins (anti-cholesterol)
      - Inhibition
        
        Reversible
        
        Non-competitive inhibition
        
        Molecule binds to allosteric site but may not influence substrate binding: 1) preventing catalysis by inactivating some enzymes (Km unaffected but Vmax decreases); 2) blocking substrate access by inducing conformational change (no change to Vmax but Km increases); 3) change substrate binding pocket by changing structure of active site (Km increase but Vmax decreases) eg antidepressants like fluoxetine and venlafaxine (monoamine oxidase inhibitors) oxidises primary amines in the enzyme (monoamine oxidase) to block metabolism of neurotransmitters.
        
        Uncompetitive inhibition
        
        Binds to enzyme-substrate complex to form ESI complex, altering the way the substrate would normally bind or dissociate, hence reducing Km and Vmax (product formation) eg Li interferes with polyphosphoinositide metabolism in brain by inhibiting inositol recycling by inositol monophosphatase (anti-mania) and Li+ ion replaces Mg2+ role as co-factor, forming salt bridge with phosphate group and preventing it from leaving the active site.
        
        Competitive inhibition
        
        Molecule (structurally similar to substrate) competes for active site but can be managed by increasing substrate conc. eg sulfonamide antibacterial drugs contains sulfuradiazine (structurally similar to para-aminobenzoic acid used to make folic acid) and binds to dihydropteroate synthase found in bacterial cells.
        
        Irreversible
        
        Significant role in drug action as it has a long term effect by permanently changing active site through direct binding or via allosteric binding, hence enzymatic activity can only be restored only after biosynthesis. Usually have features that can form covalent bonds at with amino acid residues and may carry electrophilic amino acids (to react with the nucleophilic amino acids on the enzyme surface) eg aspirin which inactivates cyclooxygenases (COX) by phosphorylating the tyrosine residues, dampening down inflammatory response. Measured in terms of Kinact. COX is involved in converting fatty acids to inflammatory molecules
- - - - Alpha helices
        
        H bonding between 1st and 4th amino acid in sequence and size are usually similar for formation of helix (disrupted by different sized amino acids). Usually terminated by proline.
      - Beta-pleated sheets
        
        H bonding between adjacent peptide chains and chains can be parallel or anti-parallel
  - - - Folding of helices to spherical structures with polar residues at surface and non-polar residues in core. Structure held together by intermolecular forces eg H bonding
    - - Coiled coil of helices or stacking of b-pleated sheets. Structure held together by intermolecular forces eg H bonding
- - - - Pro-insulin to insulin: gene encodes for pre-proinsulin and post-translational modification results in disulfide linkages in proinsulin and proteolytic cleavage occurs to form active insulin
  - - - Only occurs with glutamate molecules with addition of COO-
        group (mediated by Vit K dependent carboxylase) and important in protein activity involved in coagulation pathway
    - - Occurs only at specific amino acids (usually Aspargine-X-Serine/Threonine, where X is not Proline) with addition of sugar groups/moieties which usually changes surface properties (solubility etc). Glycosidic bonds formed between a.a and C2, C3, C4, C6 or any of the conformation isomers (axial/equatorial) at C1 of glucose (which increases variety in structures) [refer to Carbohydrates for specifics]
    - - Occurs at hydroxyl-containing amino acids and process is reversible. Usually results in formation of "anchors" to trigger binding of other proteins (biological function). Drug therapy usually inhibits phosphorylation.
    - - Tether soluble proteins to membranes for subcellular localisation to occur and polar group of lipid covalently bonded to proteins: N-terminal attachment (attached to amine group to form CONH), Thioester/thioether linkage with cysteine (attached to sulfur atom to form COS/S)
- - - - Aromatic
        
        Types: Phenylalanine, tyrosine and tryptophan
        
        Bonds: non-polar and contribute to pi-pi interactions via aromatic ring
      - Uncharged, polar
        
        Types: Serine, cysteine, methionine, threonine, asparagine and glutamine
        
        Bonds: Hydrogen bonding and disulfide bonds (cysteine)
      - Non-polar aliphatic
        
        Types: glycine, alanine, valine, leucine, isoleucine and proline
        
        Bonds: non-polar interactions and essential in stabilising folded conformation
      - Charged, polar
        
        Bond: ionic bonding or ion-dipole interactions
        
        Types: aspartic acid and glutamic acid; lysine, histidine and arginine (all are charged at pH 7)
  - - - examples:
        1) histidine
        2) isoleucine
        3) leucine
        4) lysine
        5) methionine
        6) phenylalanine
        7) threonine
        8) tryptophan
        9) valine
    - - examples:
        alanine
        asparagine
        aspartate
        arginine
        cysteine
        glutamate
        glutamine
        glycine
        proline
        serine
        tyrosine
        
        Role of cysteine
        
        proper folding and maturation of proteins (tertiary structure)
        
        Regulate oxidation process (as reducing agent)
        
        binding and quenching of reactive species as nucleophile
        *Thiol group responsible for such properties, hence cysteine derivatives are used as drugs
        
        N-acetylcysteine
        1) mucolytic agent: break down disulfide bonds in mucus proteins, reducing cross-linkages between peptide chains, reducing stickiness and viscosity. (eg fluimucil)
        2) Used as treatment of paracetamol poisoning: readily binds to electrophilic site of toxic intermediate at the aromatic ring, hence rendering it less reactive towards other proteins and increase solubility in water (for excretion).