Enzyme Kinetics

Enzymes Part 2 - Kinetics

Application of Km and Vmax: Glucokinase and Hexokinase

  • Hexokinase (most tissue) contrasts w/ glucokinase (liver) by the following:
    • Hex has lower Vmax, lower Km, greater affinity for glucose
    • Turned off by high concentrations of glucose 6-P
  • Both HexoK and GlucoK catalyze Glucose → Glucose 6-P
  • Glycolysis breaks down glucose
  • Glycogenesis stores glucose
  • Glucokinase higher Km tells us it has lower affinity for glucose, compared to hexokinase
  • Hexokinase is active during fasting
  • Greater affinity, can convert even small amounts of glucose into energy
  • Glucokinase only becomes active after a high-carb meal
  • Lower affinity, needs a lot of glucose to be activated
  • Main role in glycogenesis because you want to store this excess
  • Liver
  • Where GK is found
  • Nutrients absorbed from intestines go here first; GK converts excess glucose from a meal to glycogen
  • Other tissues cant convert excess glucose to glycogen because Glucose 6-P inhibits Hexokinase (product inhibition)
  • Hexokinase only works until you have made enough G6-P to serve body’s energy needs
  • HK feeds into glycolysis; explains low Km because even though you have little glucose, HK binds to it because it has a greater affinity
  • Makes sense that GK has a higher Km because we do not want it storing glucose unless we have already satisfied our energy needs
  • HK is inhibited by G6-P because it is mediating glycolysis; once you’ve made enough G6-P to fulfill you energy needs, HK turns off and GK turns on to store as glycogen for later use. Once glucose levels fall, GK turns off because it has a low affinity for glucose
  • Vmax
  • High Vmax = high capacity to turn substrate into product
  • Allows GK to phosphorylate glucose to glucose 6-P after a meal to store as glycogen
  • Adding P to glucose means it gets trapped in the cell
  • Km
  • Helps determine enzyme efficiency
  • How well it binds (affinity) and how quick it converts to product
  • Kcat/Km
  • Kcat measures speed of product formation once ES has been made
  • Km measures binding affinity of E and S to make ES
  • E + S
  • First you have E and S bind, then you spit out product
  • Larger value of Kcat and smaller value of Km, the greater efficiency
  • Greater speed of product formation and higher affinity
  • Enzyme efficiency
  • Larger value of Kcat/Km = more efficient
  • Enzymes in body are close to catalytic perfection; very efficient
  • Carbonic anhydrase – bicarbonate buffer
  • Triose phosphate isomerase – glycolysis
  • Fumarase – CAC
  • Acetylcholinesterase – muscle contraction; breaks acetylcholine
  • Enzyme inhibition
  • Physiological feedback mechanisms
  • Ex. Turning off HK when Glucose 6-P is too high
  • Clinical therapies
  • Use of pharmaceuticals to inhibit viral enzymes from replicating in HIV
  • Types:
  • Reversible
  • Competitive
  • Reversible binding of inhibitor to active site of enzyme
  • Ex. Methotrexate used for chemo
  • Inhibits enzyme that helps convert folate B9 into its coenzyme form*
  • Folate coenzymes normally feed into purine/pyrimidine production used to make DNA
  • Helps prevent spread of cancer cells by inhibiting DNA from replicating cell; puts break on ALL cell division
  • Vmax doesn’t change; affinity appears worse in presence of inhibitor (higher Km) Kmapp = Km apparent
  • If you go high enough in your enzyme concentration in the presence of a competitive inhibitor, you basically shove it out of the way
  • Uncompetitive
  • Reversible binding of inhibitor to ES; binds in one place
  • Rare - Inhibitor binds enzyme after substrate has bound
  • Vmax is lower with inhibitor; affinity of enzyme increases
  • You don’t get up to the same speed with an inhibitor no matter how much of the substrate you put in, because the inhibitor can always bind no matter how much substrate you have
  • Appears affinity increases because you have 2 chances for E & S to be bound
  • Noncompetitive
  • Reversible binding of inhibitor to E or ES
  • Ex. Product inhibition – G6-P inhibition of hexokinase
  • Can bind in 2 places: before the substrate does, or after
  • Always option for inhibitor to bind, no matter how much substrate
  • Lower Vmax, Km (Ki in this case) doesn’t change
  • In sum:*
  • Competitive: V max doesn’t change, affinity worse
  • Uncompetitive: Vmax is lower with inhibitor, affinity better
  • Noncompetitive: Vmax lower with inhibitor, affinity doesn’t change
  • Binding to E appears to lower affinity
  • Binding to ES appears to increase affinity