Glycolysis I

Q kinetics of biphosphate aldolase

-Q is the equil constant that considers the actual physiological concentrations, a more accurate 𝛥G than a 𝛥Gº under standard state 1M concentrations of products/reactants -actual Q depicts a -𝛥G, reflecting the favorable breaking of the fructose molecule

main reaction mechanism of fructose biphosphate aldolase

-a reverse aldol condensation reaction -aldolase cleaves 6 C fructose biphosphate into two 3 C units -must form an aldehyde on bottom half, stabilized in 2 different mechanisms (Class I and II)

class II fructose biphosphate aldolase mechanism

-cleavage product with carbonyl (not the aldehyde G-3-P product) is stabilized through its resonance structure by the lewis acid Zn2+

what is the overall 𝛥G of phosphorylation of glucose? what are the components of this rxn?

-coupling the energy intensive phosphorylation of glucose with the downhill ATP hydrolysis to get an overall favorable (-𝛥G) reaction -far removed from equilibrium

what is the purpose of triose phosphate isomerase? is it near equilibrium?

-equilibrates between DHAP (and G-3-P products from biphosphate aldolase -maintains flux of the aldose, the only structure that continues into the glycolysis pathway -near equilibirum (structures are very similar), no regulation required

what is the overall glycolysis reaction?

-glucose being oxidized to 2 pyruvate, 2NAD+ being reduced to 2 NADH, 2 ADP hydrolyzed to 2 ATP -an overall catabolism process, yields energy

how does phosphate relate to glycolysis? what does this do?

-intermediates are all bound to - charged phosphates on the 6' glucose C (glucose→glucose-6-phosphate) -this traps the intermediates into the cell, commits them to the rxn and in the cell (no electrochem gradient for it to leave)

class I fructose biphosphate aldolase mechanism

-lysine N attacks carbonyl C of fructose, creating an e- sink that helps stabilize structure once cysteine deprotonates O to create - carbonyl during cleavage

what's interesting about the ATP investment of glycolysis?

-phase 1 requires 2 ATPs invested, phase 2 yields 4 ATPs -signifies that glycolysis began from the last step to the first step (retrograde)

what are the energetics of phosphofructokinase?

-similar to hexokinase: phosphorylation of fructose on 1' C is energy intensive while coupled with favorable ATP hydrolysis -similar -𝛥G far from equil -a key regulatory step of glycolysis, the 2nd investment of ATP (ensure that we absolutely must go through glycolysis, as we will waste another ATP)

what is Q?

-the equilibrium constant with actual metabolic cell concentrations factored in (not at standard steady state) -products over reactants ex. phosphorylation of glucose: Q = [ADP][glucose-6-phosphate]/[ATP][glucose]

what are the kinetics of phosphoglucoseisomerase?

-the isomerization of glucose from an aldose to a ketose (fructose) is close to equil (glucose/fructose structures are similar to each other) -no regulation, changes in concentration can easily reverse this isomerization

triose phosphate isomerase mechanism (from DHAP to G-3-P)

-this is the +𝛥Gº, so the opposite direction is actually favored at equilibrium -so, hexoses are converted into 2 G-3-P's to get the aldoses required for continuation of glycolysis

how does a reaction being far from equilibrium relate to regulation?

-when a rxn is far from equil, this means it's a steep downhill and once the rxn starts there's no return/reverse -thus, it's important that the cell is absolutely sure it wants to commit to this rxn before starting it...so it must be regulated -the opposite is being close to equil, where the rxn can be reversed just by changing concentrations

what is the activator regulation of phosphofructokinase?

1. AMP: has one phosphate and thus is a low energy state, we need to synthesize ATP 2. fructose-2,6-biphosphate: analog of substrate, a signaling molecule that boots out the ATP attempting to bind at secondary allosteric site and inhibit phosphorylation, ↑ F-2,6-BP with ↑ ATP will still keep an michaelis curve by relieving ATP's attempted allosteric inhibition

what is the inhibitory regulation of phosphofructokinase?

1. ATP: product of glycolysis, if we have a lot of ATP why must we go through the entire glycolysis again? 2. citrate: intermediate in TCA cycle, a signal of high energy state, don't need to go through glycolysis anymore -allosteric enzyme (sigmoidal curve with ↑ conc of ATP-extra ATP binds to secondary site other than active site and supresses active site binding...normal michaelis menten curve with ↓ ATP conc)

what is the mechanism of glucose isomerization?

1. mutarotation by opening of the glucose ring 2. deprotonation of 2' C, regeneration of OH from carbonyl on 1' C 3. -ene intermediate, break the C=C by protonation of 1' C and deprotonate the 2' C to create a carbonyl now at the 2' (now an isomer, glucose→fructose!)

what enzyme catalyzes the phosphorylation of glucose?

hexokinase

where does glycolysis occur? what about the TCA cycle?

glycolysis: cytosol TCA: mitochondria

what's the significance of the muscle vs liver enzymes?

muscle: Km of 0.1mM, low Km means it can easily push glucose through to Vmax to satisfy its job liver glucokinase: Km of 10mM, needs much more substrate to react (subsaturating most of the time) and thus is more selective to responding to changes (monitors glucose, store or utilize?) -kinetic constants tailored to their functions

graphical relationship between ATP and fructose-2,6-biphosphate

↑ATP, ↓F-2,6-BP: low velocity, ATP binds to secondary allosteric binding site and inhibits ↑ATP, ↑F-2,6-BP: high velocity, analog kicks ATP off of secondary allosteric binding site, relieving inhibition and thus activates