Enzyme Regulation - DOC by 31sY3c3


									                                   Enzyme Regulation
                                   Enzyme Regulation
• Constitutive versus regulated enzymes
• 2 types of regulation
   – Fine tuning: regulation of enzyme activity
   – Course tuning: regulation of amount of enzyme made
       • Transcriptional and translational level
• Regulation deals with abundance (concentration) of substrate or product in cell
   – When product is abundant or substrate is less abundant enzyme is less active

                                      Fine Adjustment
• Postranslational regulation or regulation of activity
   – Reversible versus irreversible
   – Competitive versus noncompetitive (allosteric)
• Feedback inhibition (allosteric, reversible, noncovalent)*
   – Pathways with many intermediates
   – Final product of pathway inhibits initial step
   – When inhibitor (end product) binds to allosteric site*enzyme changes
     shape substrate doesn’t bind
                           Allosteric Enzyme
                      Feedback Inhibition In General
                           Branched Pathways
• When 2 end products result from same starting material
   – Initial enzymes are different even though starting material is the same.
   – Two initial enzymes regulated independently
• Same reaction but different regulatory control
• Independent inhibition of same enzyme by 3 products (affects the function of the isozyme)
• Example
   – Excess TYR will reduce the activity of the enzyme
   – Excess TYR and PHE will reduce the activity even more
   – When all 3 products in excess the enzymes is completely inactive

                                      Fine Adjustment
• Covalent modification
   – covalent binding of a molecule
   – Organic portion of enzyme removed
   – Reversible or irreversible
        • Reversible is more commonWhy?
   – AMP, ADP, PO4--, methylation
• How does covalent modification differ from allosteric inhibition?
                                    Glutamine Synthetase
                                     Course Adjustment
• Transcriptional versus translational regulation
   – Which mechanism is more efficient?
   – Regulation at TRC and TRL level is slower overall
• Promoter “strength” can assist in regulation
   – Strong versus weak promoters
        • Consensus and ó factor binding
             – If consensus sequence at ó factor binding site is highly conserved then sigma factor is more likely to bind
               (better complimentation)
                  » Stronger promoter
   – Proteins that are needed all the time have stronger promoters
• Operon Model
   – Synthesis of enzyme (induction) or lack of enzyme synethsis (repression)
                                             Operon Model
• Operon
   – Promoter: transcription initiation
   – Operator: stop or go signal
   – Gened are located downstream
• Operator is regulation control switch
• Regulators bind to operators to control trc
• Effectors control whether regulator will bind or not
           Course Adjustment: Transcriptional Level
Negative control mechanisms (involves stopping TRC)
• Repression*
   –   When product occurs at an excess concentration inside cell
   –   Final product (or suitable analog) represses transcription
   –   Typical with amino acid biosynthetic enzymes
   –   EX arginine synthesis
   –   Specific repression only enzymes coded within that operon are affected
• Induction*
   –   Synthesis of an enzyme only when the substrate is present
   –   Substrates or analog induces transcription of enzymes
   –   Typical with catabolic enzymes
   –   EX â galactosidase used to metabolize lactose
   –   Specific induction
 Repression of genes that code for Arginine Synthesis enzymes
                                    Specific Repression

• Regulator is a repressor protein in both cases
   – Allosteric protein that can bind to operator to repress transcription of genes downstream
     from operator
• Effectors
   – Molecules that initiate the events of repression or induction
      • Repression effectorco-repressor
          – Corepressor binds to repressor repressor can bind to operatorrepresses transcription*
          – In our example arg acted as the co-repressor
      • Induction effectorinducer
          – Inducer binds to repressor repressor leaves operatorinduces transcription*
          – In our example Lactose acted as the inducer

               Positive Transcriptional Control Mechanism
• Regulator
   – Activatorpromotes binding of RNA polymerase to DNA at promoter region
   – Binds to activator binding site
• Effector
   – Inducer must bind to activator first
   – The substrate is frequently the inducer
• EX) maltose regulon
   – Regulon: When more than one operon is under the primary control of same activator
     (regulatory) protein

                             Global Control Systems
• Starvationstimulates transcription of many genes
   – Stimulon: all the genes that are transcribed in response to such a signal
• Diauxic growth and preference for glucose
   – Glucose effect
• Example of global control: Catabolite repression modulon
   – CAP activates many catabolic pathways (alternatives to glucose catabolism) when glucose
     not available
                                       Diauxic Growth
                Lac Operon as Example of Catabolic Repression
• Catabolic repression occurs when all other metabolic pathways are repressed
  when gluc is available.
• When glucose is used up the other pathways are activated
• Lac operon really has 2 levels of regulation!
   – Lac induction (learned earlier)
   – Catabolic repression
• Glucose/Lactose media
   – When lactose present it binds to repressor and induces trclactose catabolism genes
     should be transcribed
   – Lac catabolism genes are really only transcribed if CAP protein bound to DNA as well.
      • Catabolic activator protein

                  Catabolic Repression Mechanism
• When glucose runs out an alarmone called cAMP is produced
• cAMP binds to CAP and CAP binds to DNA
   – At activator binding sites of alternative pathway metabolic enzymes (i.e. lac operon)
   – CAP activates the trc of the lac genes
• When glucose available catabolic repression will prevent organism from
  producing lactose catabolism genes because cAMP synthesis is inhibited by
  presence of glucose (i.e. no cAMP then no CAP binding)
                                      E. coli in glu and lac media
                              DNA Binding Proteins
• Sequence specific versus non sequence specific DNA binding proteins
   – Non sequence specific: histones (+ charge) keep other things from binding
   – Sequence specific: a.a. side chains interact with nucleic acids
      • Frequently occur in dimers (2 identical polypeptides)
      • Domains: the regions that actually interact with DNA
      • Regulatory proteins are all sequence specific!!!

                   DNA Binding Protein
        Transcription/Translation Level Regulation
• Premature termination of transcription results in incomplete mRNA
• Tryptophan operon regulated by repressor protein (negative control)
  and also by attenuation
   – When Trp plentiful leader translated early termination of transcription
   – When Trp lacking leader not fully translatedtrc of trp operon continues
                             Trp Operon
                   Attenuation: Tryptophan Operon
• Inverted repeat in mRNA of trp operon
   – Stem loop forms
   – 2 conformations: 3,4 stem loop and 2,3 stem loop
   – Position of ribosome
• Excess Trp
   – Leader sequence transcribed and translated fully
   – Leader polypeptide attached to ribosome
   – Causes 3,4 stem loop conformation, which signals transcription pause site and
     transcription terminated
• Trp concentration low
   – Leader sequence transcribed and translated until trp-rich region is reacheda pause occurs
   – Pause causes 2,3 stem loop conformation, which is not a termination signal and
     transcription of trp genes continues
                          Attenuation: Trp Regulation
                               Quorum Sensing
• Regulatory pathways that are controlled in response to density of cells within
• These cells secrete homoserine lactone (an inducer)
• With elevated HSL levels transcription of certain genes is induced
• EX) Vibrio fisherii, a fish symbiont, and Lux genes

                                    Signal Transduction
• 2 component Regulatory system
   – Effector molecule not always transported in
• 2 components
   – Sensor protein (in membrane)
       • Kinase activity: autophosphorylation and phosphorylation of response regulator
   – Response regulator (in cytoplasm)
       • DNA binding protein that regulates transcription
       • Must be phosphorylated to bind to DNA
• Signal must be stoppedphosphatase enzyme
   – Dephosphorylates the response regulator
• Example
   – NarX (nitrate) and NarQ (nitrite) sensor proteins
   – NarL and NarP response regulators activate transcription of nitrate and nitrite utilization genes

        Signal Transduction: 2 Component System

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