Enzymology

• There are several main types of enzymes: oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases.
• 80% of enzymatic reactions have two substrates and two products (T/F)


  False, 60% of enzymatic reactions have two substrates and two products
• what are the two mechanisms for two enzymes to react to form two products?

  1. Substituted enzyme complex

  2. Ternary complex
• what is the mechanism of the ternary complex reaction?

  1. enzyme reacts with substrate A, and then EA reacts with B to form EAB

  2. EAB makes the products, turning into ECD

  3. the first product comes off, leaving with EC, and then the second product comes off, and the enzyme is unchanged
• what is the mechanism for the substituted enzyme complex?

  1. Substrate A reacts with E, forming an EA complex

  2. the enzyme catalyses the reaction of A to C, and C leaves, but chemically modifies the enzyme

  3. the chemically modified enzyme (E), reacts with substrate B (EB), and then becomes ED

  4. D breaks off from E, and the enzyme is unchanged
• what is an example of a ternary complex enzymatic interaction?

  1. enzyme reacts with NADH and pyruvate, NADH binds first, and then pyruvate

  2. Lactate is formed, and dissociates from the complex, and NAD+ also dissociates after

  
  

  -conversion of pyruvate to lactate

  -the order of reactants is compulsory for this mechanism
• what is another example of a ternary complex enzymatic interaction?

  -conversion of creatine to phosphocreatine

  
  

  -creatine and ATP react to form phosphocreatine and ADP in the presence of an enyzme

  
  

  -the order of reactants to form the ternary complex is random in this reaction
• what is a good example of enzymes that have a substituted enzyme mechanism?

  transaminases
• what is an example of a substituted enzyme mechanism?

  1. aspartate reacts with the enzyme, and forms oxaloacetate, leaving its amino group on the transaminase

  2. alpha-ketoglutarate reacts with the modifed enzyme, recieves the amino acid, and become glutamate
• what on a lineweaver burke plot indicates a ternary complex mechanism?

  converging lines
• what on a lineweaver burke plot inciates a substituted enzyme mechanism?

  parallel lines
• what are the three typies of reversible inhibition?

  1. competitive

  2. uncompetitive

  3. non-competitive
• what are the two types of inhibition?

  1. reversible

  2. irreversible
• how can you tell if inhibition is irreversible?

  inhibitor cannot be removed by dialysis or filtration

  
  

  -inhibitor forms a covalent bond with an amino acid sidechain at the active site
• how can you tell if inhibition is reversible?

  -inhibitor can be removed by dialysis or filtration
• how does dialysis work to determine reversible vs. irreversible?

  1. place the inhibitor inside a dialysis set with a semi-permeable membrane

  2. small molecules will equilibirate through the membrane, while proteins are too large to move across the membrane

  3. if the mechanism is reversible, the inhibitor will come out of the dialysis set

  .4 if the mechanism is irreversible, the inhibitor will not come out into the solution of the dialysis set
• what are four examples of irreversible inhibitors?

  1. iodoacetic acid

  2. diisopropylphosphofluoridate

  3. tosyl-L-phenylalanine chloromethyl ketone (TPCK)

  4. suicide inhibitors
• iodoacetic acid inhibition MoA


  -an enzyme with an -SH group on the active site is involved

  1. the hydrogen proton comes off of the SH group

  2. iodoacetica acid reacts with the SH group, forming a covalent bond and shedding iodine
• diisopropylphosphofluoridate MoA inhibition


  an enzyme with an -OH group forms a covalent bond with the phosphate, losing a fluorine ion
• what does diisopropylphosphofluoridate act on?

  AChesterases
• TPCK MoA inhibition


  -reacts with a histidine at the active site of chymotrypsin, as the phenylalanine mimicks specificity, binds and reacts at the active site
• what is the difference in selectivity for TPCK and TLCK?

  TLCK: tosyl-L-lysine chloromethyl ketone

  
  

  -TLCK is an irreverislbe inhibitor of trypsin

  -TPCK is an irreversible inhibitor of chymotrypsin
• what are two examples of suicide inhibitors?

  1. Deprenyl

  2. fluorouracil
• what is a suicide inhibitor?

  binds to the enzyme as a normal substrate does, reactive intermediate is generated, which reacts with a group at the active site, inhibiting the enzyme
• fluorouracil MoA


  -used in cancer chemotherapy

  
  

  -inhibits thymidylate synthase when it is converted to fluorodeoxyuridate

  
  

  -when fluorodeoxyuridate reacts with cofactor methylene tetrahydrofolate an thymidylate synthase, the reactive intermediate generated is stable, and the enzyme is inhibited
• what is the normal reaction that thymidylate synthase undergoes?

  converts dUMP to DTMP, a covalent intermediate between the dUMP, cofactor methylene tetrahydrofolate, and a thiol group at the active site was formed
• deprenyl MoA


  -inhibits monoamine oxidase, used in the treatment of Parkinson's and depression

  
  

  -deprenyl binds to monoamine oxidase, which has a tightly bound FAD cofactor

  -the flavin oxidises deprenyl, and then the oxidised deprenyl reacts with the flavin, inhibiting the enzyme
• where are the two points that can be inhibited in reversible inhibition?

  before the substrate binds to the enzyme, or it can bind to the enzyme substrate complex
• what is the equilibrium constant for binding to the enzyme before the substrate?

  K_I= ([E][I])/[EI]
• what is the equilibrium constant for binding to the enzyme-substrate complex?

  K_I'= ([ES][I])/[ESI]
• where does a competitive reversible inhibitor bind?

  at the enzyme active site, directly blocking the binding of the substrate
• where does a non-competitive reversible inhibitor bind?

  non-competitive inhibitors do not prevent the substrate from binding, but no product is formed as the substarte cannot bind properly
• what does the lineweaver burke plot of competitive inhibition look like?

  1. differing slopes

  2. the graph converges at the y intercept, which means that V_max of the enzyme remains the same

  
• what are qualities of competitive inhibition in terms of V_max, K_m

  1. K_m increases because more [S] is needed to achieve the previously observed V_max

  2. V_max is unchanged
• what is an example of reversible competitive inhibition?

  benzamidine binding to trypsin--mimicking arginine
• what does the lineweaver burke plot of non-competitive inhibition look like?

  1. differing slopes

  2. converge at K_m, e.g. the x-axis
• what can a non-competitive inhibitor bind to?

  the enzyme with or without substrate, so E or ES
• what are qualities of non-competitive inhibition in terms of V_max, K_m

  V_max decreases--active molecules of enzyme are removed

  
  

  -K_m remains the same, as the inhibitor does not interfere with the binding of the substrate to the enzyme
• what can an uncompetitive inhibitor bind to?

  enzyme substrate complex
• what does the lineweaver burke plot of uncompetitive inhibition look like?

  1. slope is unchanged

  2. K_m decreases -equilibria shifts towards ES and ESI complex formation

  3. V_max decreases -active ES molecules are removed

  4. lines do not converge.