1b Glucose Catabolism: Control of Glycolysis (Slides 54-70)

1b Glucose Catabolism: Control of Glycolysis (Slides 54-70)

1. Under "steady state" conditions, glycolysis operates continuously in most tissues

2. Reactions 1 (hexokinase), 3 (phosphofructokinase), 10 (pyruvate kinase) have very negative delta-G's and, for that reason, are candidates for the flux control points in glycolysis. [As shown in the figure on slide 56] The other 7 reactions operate at or around delta-G = 0 and are concentration dependent. These 7 reactions readily accommodate changes from flux controlled points.

   • 1. Hexokinase (slides 6-7)
     • 1st step catalyzed by hexokinase: Glucose + ATP → G6P + ADP + H+
     • Transfer of high energy phosphoryl group from ATP to glucose
     • Mg+2 cofactor
       • Mg+2 complexes with ATP forming Mg+2-ATP complex
         • ATP is a competitive inhibitor to stop hexokinase
     • Non-specific - Hexokinase phosphorylates glucose, fructose & mannose, but not galactose
     • Glucose induces huge conformational change in hexokinase
       • 2 lobes swing together trapping glucose in active site & excluding water
         • Places ATP close to #6 –OH of glucose
           • Catalysis by proximity
             • If water present – it reacts with ATP >>>>
     • Location: everywhere except liver and kidneys (glucokinase in those places)
   • 10. Pyruvate Kinase (slides 34-5)
     • [other slide show]
   • 3. Phosphofructokinase (slides 15, 54-70)
     • Major control point for glycolysis in muscle under most conditions
     • It is a tetramer with 2 conformations T and R
       • ATP is both substrate and allosteric inhibitor
         • Each phosphofructokinase subunit has 2 binding sites for ATP
           • ATP substrate site – binds ATP equally in either T or R
           • ATP inhibitor site – binds ATP exclusively in T state-->
           • F6P binds preferentially R state
         • ADP, AMP and F2,6P are activators by reversing the inhibitory effect of ATP
     • When there is High [ATP]
       • Leads to shift to T state and decrease of phosphofructokinase’s affinity for F6P, which shuts down phosphofructokinase by it not binding to F6P
       • <-- ATP acts as allosteric inhibitor of phosphofructokinase by binding to T state
         • Allosteric site has larger km than substrate binding site
     • Graph of phosphofructokinase activity vs. [F6P] --- Figure on Slide 60
       • With low ATP or no inhibitors, phosphofructokinase activity near maximum
       • As [ATP] increases, it shifts the kinetics curve to the right (same VMax, but KMs decrease as you increase ATP)
         • Becomes more sigmoidal (more cooperativity)
         • Activators, such as AMP, counter ATP effect by binding to R state of phosphofructokinase (shifting equilibrium to R state)
     • Direct allosteric control of phosphofructokinase by ATP appears to be all that is needed to control glycolysis flux
       • When [ATP] is high due to low demand of ATP, phosphofructokinase is inhibited and glycolysis flux is low
       • When [ATP] is low, glycolysis flux is high to replace ATP
       • Flux thru glycolysis varies by 100x or more (due to AMP changes)
         • Results from preferential binding to R state
         • [ATP] varies < 10% between rest & exercise due to due to buffering activity of (1) Creatine kinase (ATP + creatine ←→ creatine~P + ADP) and (2) Adenylate kinase
           • Adenylate kinase catalyzes 2 ADP ←→ ATP + AMP
             • K = [ATP][AMP]/[ADP]2 = 0.44
           • This rapidly equilibrates [ADP] resulting from ATP hydrolysis in muscle contractions
             • In muscle: [ATP] ~ 50X [AMP] & ATP ~ 10X [ADP]
               • ∴ 10% ↓[ATP] results in 100% ↑[ADP] as result of adenylate kinase & > 400% ↑[AMP]
               • ∴ metabolic signal ↓ in [ATP]:
                 • Too small to relieve phosphofructokinase inhibition (or increase enzyme activity)
                   • AMP accounts for activation of phosphofructokinase (overcoming of inhibition) K = 0.5
                 • But is amplified significantly by adenylate kinase reaction which ↑ [AMP] by amount producing much larger ↑ in phosphofructokinase activity
                 • 2 ATP + 2 creatine ←→ 2 creatine~P + 2 ADP ←→ ATP + AMP
                   • [ATP]↓ pulls equilibrium to right: [ADP]↑ & [AMP]↑
     • Substrate Cycling
       • As noted earlier, allosteric control can not account for 100X change in glycolysis flux --->
       • GENERAL: 2 different enzymes catalyze forward & reverse reaction and can be independently varied in a thermodynamically favorable manner
         • F6P + ATP → FBP + ADP (ΔG = -25.9kJ/mol)
           • 2 opposing reactions cycling substrate to an intermediate and back
             • Combined opposing reactions produce much greater pathway flux than possible with allosteric regulation of single enzyme
               • Cycling appears to be “energetic price” (ATP) muscle pays to be able to switch from resting to maximum activity
                 • Can turn on one enzyme while we turn off the other (one for glycolysis with the other in gluconeogenesis
                   • Controlled by level of F2,6P (controlled by cyclic AMP)
         • In gluconeogenesis, fructose-1,6-bisphosphatase (FBP)
           • FBP + H2O → F6P + Pi (ΔG = -8.6 kJ/mol)
             • combined reaction: ATP + H2O ←→ ADP + Pi
       • SPECIFIC: Thermogenesis = Generate body heat thru substrate cycling in liver and muscle (nonshivering thermogenesis)
         • stimulated by thyroid hormones (which also stimulate metabolism)
         • Chronically obese tend to have lower metabolic rates & tend to be more cold sensitive – lower rates of nonshivering thermogensis
         • Muscle contractions of shivering also produces body heat