Altering enzyme activity

• Factors affecting enzyme activity

  • Inhibitors
  • Physical factors
  • Cellular regulation
• How does temperature affect enzyme activity

  
  

  1. As temperature increases, collisions between substrates and active sites occur more frequently as molecules move faster

  
  

  2. Thermal agitation disrupt the weak bonds that stabilise the enzyme conformation, leading to thermal denaturation
• Effects of pH on enzyme activity

  
  

  1. pH influences enzyme conformation and electrostatic interactions between enzyme and substrate (ionisation)

  2. Optimum between pH 6–8 for most enzymes

  3. Shifts equilibrium for reactions that involve hydrogen ions
• What factors affect Enzyme activity in a cell

  
  

  • How much enzyme is present
  • The absolute activity of the enzyme present [Regulation]
• Feedback inhibition

  When the final products acts an inhibitor for the enzyme which catalyses the committed step.
• What type of regulation is used in serine in E.coli synthesis

  Feedback inhibition.

  3-phosphoglycerate is converted by 3 enzymes to Serine.

  Serine regulates the activity of the first enzyme
• Describe the structure of 3-phosphoglycerate dehydrogenase

  It is a tetramer with 4 identical subunits.

  4 catalytic and 4 regulatory serine binding sites.
• Describe the features of Allosteric regulation

  Associated with multi-subunit enzymes.

  It can be positive [cooperative] or negative [allosteric inhibition].
• Describe Positive cooperative allosteric regulation

  These enzymes are called allosteric enzymes and produce a sigmoidal curve.

  Substrate binding decreases the Km at the other active sites.
• Describe the production of the sigmoidal allosteric enzyme curve

  It is a combination of the T-state and R-state Michaelis-Menten plots.

  when the Enzyme changes from T-state to R-state Km decreases and so reaction rate increases.
• Allosteric regulation: Aspartate carbamoyltransferase (ACTase)

  • Aspartate is a positive allosteric regulator it binds to the catalytic subunit
  • CTP is a negative allosteric regulator it binds to the regulatory subunit
• ACTase structure

  It is a multi-subunit enzyme.

  The 6 catalytic subunits bind the substrates.

  The 6 regulatory subunits have no catalytic activity and is regulated by CTP.
• How was the ACTase reaction mechanism discovered

  PALA is a stable transition state analogue which forms a stable transition state and the structure was visualised.
• What type of binding is used in ACTase

  Cooperative. Substrate binding induces a conformational change.
• What reaction does lactate dehydrogenase [LDH] catalyse

  The inter-conversion of lactate and pyruvate using a NAD+ cofactor.
• Isozymes: Lactate dehydrogenase (LDH)

  • H form (heart)
  • M form (skeletal muscle)
• What is the difference between the H and M forms of LDH

  The H form has a higher affinity for pyruvate and lactase and is allosterically inhibited by pyruvate.

  It is also optimised to convert lactate to pyruvate and eventually oxygen by gluconeogenesis to provide fuel for aerobic metabolism.

  
  

  The M form has faster catalysis and is optimised to convert pyruvate to lactate for anaerobic glycolysis.
• Which metabollic pathways is Lactate dehydrogenase used in

  Glucose metabolism and syntehsis.
• What does Q10 = 2 mean

  The rate doubles for every 10C rise in temperature
• What is the effect of a high amount of product

  It may inhibit enzyme activity by product inhibition and reverse catalysis.
• What is the committed step in a biochemical pathway

  It is the slowest and often called the rate-limiting step.

  It is also the first irreversible step.
• What is the first enzyme in the serine production pathway

  3-phosphoglycerate dehydrogenase
• What happens to 3-PGDH as serine concentration increases

  Serine binds to regulatory sites, decreasing Vmax.

  When 4 serine molecules have bound it is inactive.
• What is an allosteric effect

  When binding at one site causes an affect at a distinct site
• What types of regulation are used for 3-phosphoglycerate dehydrogenase

  Feedback and allosteric.
• What type of regulation is used for aspartate carbamoyltransferase

  Allosteric
• What is the equation for the production of aspartate carbamoyltransferase [ACTase]

  Carbamoyl phosphate + Aspartate
• What is the first comitted step in pyrimidine synthesis

  Aspartate carbamoyltransferase production from carbamoyl phosphatase and aspartate
• Describe the conformation change in ACTase

  The catalytic trimers rotate and move apart this pull the regulatory units apart.

  The structure changes from the low activity T-state to the high activity R-state.
• What is the effect of CTP on ACTase

  CTP binds to the regulatory subunits and stabilises the T-state.

  The curve shifts to the right and the enzyme requires a high substrate concentration to be activated.
• What are the features of isozymes

  Different versions of the same enzyme encoded by different genes which are part of gene families.

  That catalyse the same reaction but have different catalytic properties. This includes Km, Vmax and the response to regulatory molecules.
• How is glucose borken down in muscles

  By glycolysis to pyruvate. Lactose dehydrogenase converts pyruvate to lactic acid which is removed by the blood.
• How is glucose synthesised in the liver

  Lactic acid is converted to pyruvate using lactose dehydrogenase. Then it is converted to glucose via gluconeogenesis.
• How many isoforms are there of LDH

  5
• What is the structure of LDH

  It is a tetramer which can exist in different combinations of the M and H forms.
• What happens to the isoform composition of LDH as the heart develops

  When in the womb, the heart uses the M form for the anaerobic environment and then changes to the H form for the aerobic environment after birth.