L26: Allosteric Regulation and Commercialisation of enzymes

• What is the full name of the enzyme referred to as ATCase?

  Aspartate transcarbamoylase
• Why is catalysis by ATCase considered the rate-limiting step in pyrimidine biosynthesis?

  Because it alters its catalytic activity in response to cellular levels of CTP and ATP
• What effect does CTP have on ATCase activity?

  CTP decreases activity
• What effect does ATP have on ATCase activity?

  ATP increases activity
• What happens to the curve when 0.4 mM CTP is added?

  The curve shifts to the right
• What happens to the curve when 2 mM ATP is added?

  The curve shifts to the left
• What type of kinetics does ATCase exhibit?

  Non-Michaelis-Menten kinetics
• What does increasing concentrations of CTP do to the apparent Km for aspartate?

  It increases the apparent Km for aspartate
• How can allosteric interaction of ATCase be examined?

  Through structural studies using an inhibitor called PALA
• What does PALA mimic in the study of ATCase?

  PALA mimics the reaction intermediate
• What state does PALA stimulate the formation of in ATCase?

  The R, relaxed state
• What is the activity level of the T state of ATCase?

  Low activity
• What is the activity level of the R state of ATCase?

  High activity
• What does allosteric regulation of ATCase involve?

  Equilibrium between the R and T states
• What effect does CTP binding have on ATCase?

  It stimulates an allosteric shift to the T state
• What effect does ATP binding have on ATCase?

  It stimulates an allosteric shift to the R state
• What is the key enzyme in glycolysis mentioned in the material?

  Pyruvate kinase (PK)
• What metabolic process occurs when glucose is in short supply?

  Gluconeogenesis
• What is the relationship between glycolysis and gluconeogenesis?

  They are interconnected metabolic pathways
• What is a futile cycle in metabolism?

  A cycle where glycolysis and gluconeogenesis occur simultaneously without net gain
• What is feed forward activation in the context of pyruvate kinase?

  PK is activated by an early intermediate in glycolysis, FBP
• What is feedback inhibition in the context of pyruvate kinase?

  PK is inhibited by an end product, ATP
• What is the summary of allosteric regulation?

  • Allosteric regulation makes enzymes 'switchable'
  • Binding of effectors to secondary sites alters activity
  • Effectors can be activators or inhibitors
  • Changes operate through conformational changes affecting the active site
  • Displays non-Michaelis-Menten kinetics with co-operativity
  • Regulates flux of metabolites through pathways
• What are some applications of enzymes mentioned in the material?

  Enzymes are used in farming, food industry, biosensors, and therapeutics
• Why are shikimate pathways attractive targets for antimicrobial agents and herbicides?

  They are absent in animals
• What is the mechanism of action of glyphosate as a herbicide?

  Glyphosate inhibits EPSP synthase in a competitive manner
• What is the purpose of genetic engineering in crops to be Roundup resistant?

  To fuse the bacterial gene encoding CP4 EPSP synthase to a chloroplast signal peptide
• What was the first Roundup Ready commercial crop?

  Roundup Ready soybeans
• What are some pros and cons of glyphosate-resistant crops?

  Pros include reduced tilling and increased profits; cons include lower yield and potential increased herbicide resistance in weeds
• What is the first approved GMO herbicide-resistant crop?

  Roundup Ready soybeans
• What are some sources of enzymes for food applications mentioned in the material?

  Traditional sources include intact microbes
• What are some applications of enzymes in the food industry?

  • Cheese making
  • High fructose corn syrup production
  • Aspartame production
  • Other food processing applications
• What was the yield difference of Roundup Ready soybean crops compared to conventional varieties in 1999?

  Roundup Ready soybean crops had a 6.7% lower yield.
• What are some approved GMO herbicide resistant crops and their uses?

  • Alfalfa: Animal feed (USA, approved 2005)
  • Canola: Cooking oil, margarine (USA, approved 1995)
  • Cotton: Fibre, cottonseed oil (USA, approved 1994)
  • Maize: Animal feed, high-fructose corn syrup (USA, approved 1995)
  • Soybean: Animal feed, soybean oil (USA, approved 1993)
  • Sugar Beet: Food (USA, approved 1998)
• Who delivered Lecture 26 on enzymes in the context of the food industry?

  1. Robert Spooner.
• What are the sources of enzymes for food applications?

  • Traditional: Using intact microbes (e.g., lactobacilli, S. cerevisiae, Aspergillus oryzae)
  • Increasingly: Enzymes or enzyme mixtures added to bioreactors
• What is the structure of cow's milk and its main components?

  • Water: 85.5 - 89.5%
  • Fat: 2.5 - 6%
  • Protein: 2.9 - 5% (80% casein)
  • Lactose: 3.6 - 5.5%
  • Minerals: 0.6 - 0.9%
• What role does chymosin play in milk structure?

  Chymosin cleaves κ-casein, destabilizing micelles and allowing milk to clot.
• What is the enzyme classification number for chymosin?

  EC 3.4.23.4.
• What are the benefits of using recombinant chymosin in cheese production?

  • High production yield
  • Better curd texture
  • Reduced bitterness
  • Suitable for kosher and halal certification