01 Hexokinase (HK)
Glucose + ATP --> G6P + ADP + Pi (ΔG = -16.7 kJ/mol, must be regulated.)
Transferase. Has a cofactor: Mg2+.
+ Effector: Pi
- Effector: G6P (feedback inhibition)
Mechanism: Uses ATP to phosphorylate the glucose. Keeps concentration of glucose low in the cell thereby promoting continuous transport of glucose into the cell through transporters. Also traps glucose in the cell b/c there is no transporter for G6P, & G6P cannot diffuse out due to its charge. HK has a specific type of induced-fit substrate-binding mechanism/a coupled, sequential reaction: Glucose binds HK first, excluding water from the active site, and induces a conformational change before binding ATP to prevent futile hydrolysis of ATP.
Liver contains both hexokinase and the hexokinase isozyme glucokinase; both catalyze the phosphorylation of glucose to G6P, but GK is not inhibited by G6P. The principle of "regulation by substrate availability":
1) HK will catalyze the reaction near maximal velocity at virtually all times. However, it is inhibited by G6P (its product). It is not regulated by substrate availability since energy metabolism is important even when fuel is in short supply.
2) GK has a much lower affinity for glucose than HK and is regulated by substrate availability - Velocity of catalysis increases with increasing glucose concentration. It is not inhibited by product, however. When blood glucose levels are high, GK converts excess glucose to glycogen, fatty acids, and cholesterol even as G6P accumulates (while HK is inhibited by G6P). Phosphorylation also brings excess glucose into the liver, thereby removing free glucose from the equilibrium.
High [glucose]: In the liver, extra G6P may be converted to G1P for conversion to glycogen, OR converted by glycolysis to acetyl-CoA and then citrate. Excess citrate is exported to the cytosol, where ATP citrate lyase will regenerate acetyl-CoA and OAA. The acetyl-CoA is then used for fatty acid synthesis & cholesterol synthesis, two important ways of utilizing excess glucose when its concentration is high in blood.
Low [glucose]: During hypoglycemia (low glucose), glycogen is converted back to G6P and then to glucose by the liver-specific enzyme glucose-6-phosphatase and released into the blood without taking up the low concentration of glucose it releases. This reverse reaction is an important role of liver cells to maintain blood sugars levels during fasting. This is critical for brain function, since the brain utilizes glucose as an energy source under most conditions.
HK: catalyzes near maximal velocity at virtually all times. Inhibited by product.
GK: catalyzes with increasing glucose concentration. Not inhibited by product.
1. GK will continue to catalyze the reaction for glucose --> G6P. GK converts excess glucose to glycogen, fatty acids, cholesterol.
2. Extra G6P --> G1P --> Glycogen
3. Glycolysis: Extra G6P --> acetyl-CoA --> citrate. Citrate is exported to cytosol. ATP citrate lyase: Citrate --> acetyl-CoA + OAA.
4. Acetyl-CoA --> fatty acids, cholesterol
1. Glucose-6-Phosphotase: Glycogen --> G6P --> Glucose
02 GlucosePhosphate Isomerase
G6P --> F6P (ΔG = +1.67 kJ/mol)
Isomerization of G6P to a keto sugar via an enediol intermediate is necessary for carbanion stabilization in the fourth reaction step. It primes the substrate for phosphorylation at C1 (more favorable for -OH than hemiacetal). It also activates C3 for aldol cleavage (need vicinal C=O & C-OH) in step 4 by aldolase into DHAP & GAP.
Driven forward because of a low concentration of F6P, which is constantly consumed during the next step of glycolysis. Under conditions of high F6P concentration, this reaction readily runs in reverse.
03 PhosphoFructoKinase (PFK)
F6P + ATP --> F-1,6-bisP + ADP + Pi (ΔG = -14.2 kJ/mol, must be regulated.)
Transferase. Has a cofactor: Mg2+.
+ Effectors: AMP/ADP (energy charge), Pi, NH4+, F-2,6-bisP (except in plants & bacteria), F6P - they bind preferentially to R-state and stabilize it.
- Effectors: ATP, Citrate, H+ - ATP binds preferentially as a negative effector to the regulatory site on the T-state and stabilizes it.
Mechanism: Uses ATP. Commits the Carbons of glucose to catabolism. Is the rate-limiting step of glycolysis. Is a major regulatory step. Is the second use of ATP as an intermediate phosphate carrier to achieve favorable thermodynamics.
Regulation: PFK is an allosteric enzyme with multiple positive & negative effectors. It is sensitive to energy status of the cell. It is a tetramer of 4 identical subunits and exhibits positive cooperativity only when negative effectors are bound.
Cascade Regulation: PFK-2 has 2 forms:
1) [Glucose] is high: PFK-2 is dephosphorylated --> PFK-2 has kinase activity and phosphorylates F6P to F-2,6-bisP, which is a positive effector of PFK. Insulin is a sign of abundant glucose, it stimulates PFK-2 to make F-2,6-bisP (a sign that there is a lot of glucose).
2) [Glucose] is low: PFK-2 is phosphorylated --> PFK-2P has phosphotase activity and dephosphorylates F-2,6-bisP to F6P to encourage glycolysis. Glucagon ("glucose is gone") is a sign of low glucose, and stimulates cAMP production, which acts to phosphorylate PFK-2 (becomes PFK-2P) and converts F-2,6-bisP to F6P to continue glycolysis.
F-2,6-bisP is a very potent activator of phosphofructokinase (PFK-1), and is synthesized when F6P is phosphorylated by a second phosphofructokinase (PFK-2).
In liver, when [glucose] is low: Both glucagon and epinephrine cause high levels of cAMP in the liver, and PFK-2 is phosphorylated by protein kinase A. Phosphorylation inactivates PFK-2, and another domain on this protein becomes active as F-2,6-bisPhosphatase, which converts F-2,6-bisP back to F6P. The result of lower levels of liver F-2,6-bisP is a decrease in activity of PFK-1 (does not catalyze as much F6P) and an increase in activity of F-1,6-bisPhosphatase, so that gluconeogenesis (in essence, "glycolysis in reverse") is favored. This is consistent with the role of the liver in such situations, since the response of the liver to these hormones is to release glucose to the blood.
ATP competes with AMP for the allosteric effector site on PFK. An increase in AMP is a consequence of a decrease in energy charge in the cell.
Citrate inhibits PFK by enhancing the inhibitory effect of ATP (in vitro). However, it is doubtful that this is a meaningful effect in vivo, because citrate in the cytosol is utilized mainly for conversion to acetyl-CoA for fatty acid and cholesterol synthesis.
F-1,6-bisP --> Dihydroxyacetone-P (DHAP) & Glyceraldehyde-3-P (GAP) (ΔG = +22.8 kJ/mol)
Reaction occurs because the cellular concentration of F-1,6-bisP is cleaved into 2 products at very low concentrations. Therefore, the ΔG is slightly negative under physiological conditions as long as the concentrations of the 2 products is kept low by their utilization in other biological reactions (ΔG becomes -0.38 kJ/mol, so reaction can be reversed by altering concentrations). DHAP & GAP are thereby depleted by subsequent reactions of glycolysis and utilization in other pathways; both are important metabolic pathways (such as Gluconeogenesis (GNG), Photosynthesis, phospholipid synthesis).
Reaction occurs via a "Schiff Base" covalent intermediate.
05 TriosePhosphate Isomerase (TIM)
DHAP --> GAP (ΔG = +7.5 kJ/mol)
Isomerization allows metabolism of Carbons of glucose to be completed by a single pathway.
TIM is a "catalytically perfect enzyme" - It is so efficient at binding the substrate and catalyzing the reaction that product is formed as soon as the substrate collides with its active site. So rate is limited only by the rate of diffusion and a higher Kcat would not result in a faster rate of catalysis.
It has an alpha/beta barrel structure with a flexible extended loop at the active site. TIM thus undergoes a conformational change after substrate-binding - the loop closes like a lid over the intermediate stabilizing it, enhancing the catalysis.
Interesting: Aldolase (Step 4), Enolase (Step 9), and Pyruvate Kinase (Step 10) all have the alpha/beta barrel structure. They catalyze different types of reactions but they all have their active sites at the same end of the barrel (the carboxyl-terminal end of the beta-strands), and several of them have flexible extended loops as well. Divergent evolution from a common ancestral protein? Perhaps, but there is little sequence similarity.
TIM's isomerization of DHAP to GAP is most similar to GlucosePhosphate Isomerase (G6P --> F6P), which are ketose-aldose isomers as well. (G6P was isomerized to F6P to prime it for Phosphorylation at C1 and eventual aldol cleavage at C3).
(END OF STAGE I)
06 Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH)
2(GAP + NAD+ + Pi --> 1,3-bisPhosphoGlycerate/1,3-BPG + NADH + H+) (ΔG = +6.3 kJ/mol)
Incorporates Pi, produces 2 NADH.
Incorporates inorganic phosphate into the pathway to make net synthesis of ATP possible. Phosphorylation is driven by free energy change of the oxidation-reduction. The next reaction is highly spontaneous and helps pull this reaction forward by removal of product.
07 PhosphoGlycerate Kinase (PGK)
2(1,3-bisPhosphoGlycerate/1,3-BPG + ADP --> 3-PhosphoGlycerate/3-PG + ATP) (ΔG = -18.8 kJ/mol)
Transferase. Has a cofactor: Mg2+.
Produces 2 ATP.
1,3-BPG has a higher phosphoryl than ATP, so it can transfer the phosphate. Occurs via substrate-level phosphorylation: a phosphoryl group is transferred from a metabolic intermediate to ADP to form ATP (direct phosphorylation, avoids oxidation as in oxidative phosphorylation). Because this step requires ADP, when the cell has plenty of ATP (and little ADP), this step does not occur. Because ATP decays relatively quickly when it is not metabolized, this is an important regulatory point in the glycolytic pathway.
Glycolysis has now reached the break-even point: 2 molecules of ATP were consumed, and 2 new ATP have now been synthesized.
08 PhosphoGlycerate Mutase (PGM)
2(3-PhosphoGlycerate/3-PG --> 2-PhosphoGlycerate/2-PG) (ΔG = +4.7 kJ/mol)
Isomerase: apparent transfer of functional groups.
A Histidine on the enzyme already has a Phosphate attached. This Phosphate is attached to C2 of 3-PG, and the enzyme takes the Phosphate from C3 to make 2-PG.
2(2-PhosphoGlycerate/2-PG --> PhosphoEnolPyruvate/PEP) (ΔG = +3.2 kJ/mol)
Lyase. Has a cofactor: 2 Mg2+.
Catalyzes the 2nd "high-energy" intermediate formation in glycolysis.
Mutase & Enolase reactions make the second ATP generation possible:
3-PG + H2O --> G + Pi (ΔG = -17.6 kJ/mol, not enough to drive ATP synthesis)
PEP + H2O --> Pyruvate + Pi (ΔG = -61.9 kJ/mol, more than enough to drive ATP synthesis, when added to a ΔG = +30.5 kJ/mol for ADP + Pi --> ATP in next step!)
10 Pyruvate Kinase (PK)
2(PEP + ADP --> Pyruvate + ATP) (ΔG = -31.4 kJ/mol, must be regulated)
Produces 2 ATP via substrate-level phosphorylation.
+ Effector: AMP/ADP, F-1,6-bisP, or (F-2,6-bisP, a regulator of PFK).
- Effector: ATP, Acetyl-CoA, NADH (NADH leads to ATP), Alanine (leads to Pyruvate), long-chain fatty acids (leads to acetyl-CoA) --> all 3 are signs of abundant energy, so they will inhibit PK.
Other regulation mechanisms: L-isozyme is also regulated by reversible phosphorylation. Phosphorylated form is inhibited, dephosphorylated form is active.
1. Liver PK is also regulated indirectly by epinephrine and glucagon, through protein kinase A (same as PFK). This protein kinase phosphorylates liver PK to deactivate it.
2. Muscle PK is not inhibited by epinephrine activation of protein kinase A.
3. Glucagon signals fasting (no glucose available). Thus, glycolysis is inhibited in the liver but unaffected in muscle when fasting.
High [glucose]: An increase in blood sugar leads to secretion of insulin, which activates phosphoprotein phosphatase I, leading to dephosphorylation and activation of PK. These controls prevent PK from being active at the same time as the enzymes that catalyze the reverse reaction (pyruvate carboxylase and phosphoenolpyruvate carboxykinase in gluconeogenesis), preventing a futile cycle.
What is the net effect of Glycolysis?
Input of 2 ATP
Produce 4 ATP (from PhosphoGlycerate Kinase & PK reaction)
For a net of 2 ATP (from PK reaction) + 2 NADH (from GAPDH reaction)
Net reaction of Glycolysis: Glucose + 2 ADP + 2 Pi + 2 NAD+ --> 2 Pyruvate + 2 ATP + 2 NADH + 4 H+ + 2 H2O
GAPDH & Phosphoglycerate Kinase are complexed
Product is handed off directly from GAPDH to Phosphoglycerate Kinase.