Glycolysis 1
What are the two phases of glycolysis, and what are the products of glycolysis?
The two phases are: Investment and payoff phase (or preparative and ATP-generating phase). You must invest 2 ATP to activate glucose, and 4 ATP are generated in the payoff phase. Net product is 2 ATP. We also produce 2 pyruvate and 2 NADH in glycolysis.
What is the function of glycolysis? In what metabolic state would this be most active? In what two organs is this always occurring?
Function: To provide energy to the cell.
Metabolic State: Fed state (insulin stimulates)
Organs: RBCs, brain
Fructokinase/essential fructosuria.
A deficiency in fructokinase will not allow fructose to be metabolized (fructose cannot be converted to fructose 1-phosphate), resulting in elevated serum fructose after eating a meal containing sucrose or fructose. The elevated fructose leads to no metabolic problems, but does enter the urine for excretion. Because fructose is a reducing sugar (as is glucose), it will react positively in a glucose dipstick test. A more specific glucose oxidase test would need to be run to demonstrate that the positive result on the dipstick test was or was not due to elevated glucose.
How does galactose provide us energy? (I.e. what product in the glycolytic pathway is it converted into?)
G6P
Classical: Gal-1-P uridylyltransferase
NOnclassical: GalactOkinase
What are the three important steps in glycolysis?
Hexokinase/glucokinase, PFK-1, and pyruvate kinase
What enzymes use ATP in the glycolytic pathway, and what enzymes produce ATP?
Enzymes that produce ATP: Phosphoglycerate kinase, pyruvate kinase
Cilostazol is a cAMP phosphodiesterase inhibitor that is used in the treatment of peripheral artery disease. Theoretically, what effects could this drug have on pyruvate kinase activity in the liver?
Recall that glucagon inhibits pyruvate kinase in the liver (think we don't want glycolysis happening when we are undergoing gluconeogenesis). Glucagon activates the Gs pathway, which results in PKA activation. PKA phosphorylates and inactivates pyruvate kinase, which would inhibit the function of this enzyme and glycolysis.
Galactose is first phosphorylated by galactokinase to yield gal-1-P. Then, it is converted into glucose-1-P. This occurs via galactose-1-phosphate uridylyltransferase, which swaps UDP-glucose for UDP-galactose. (The UDP-galactose can then be epimerized into UDP-glucose and the cycle can be repeated). Anyway, then G-1-P can be isomerized into G6P.
Which galactosemia could be considered worse? Why?
What is the difference between hexokinase and glucokinase? Why is this important?
Hexokinase is expressed in RBCs. It has a very low Km (high affinity for glucose), so it ensures that RBC's always are utilizing glucose for energy, as it is their only source of fuel. Glucokinase, expressed in the liver, has a Km near the glucose concentration of the serum. When glucose concentration increases (like after a meal), it increases the conversion of glucose to G6P in the liver.
Also: hexokinase is product regulated but glucokinase is not.
What enzyme produces NADH in glycolysis?
GAP dehydrogenase
In the cardiac isozyme of PFK-2, Protein Kinase B phosphorylates a regulatory serine site on the F-2,6-BPase domain. What activates Protein Kinase B, and what effect would this have on glycolysis?
By phosphorylating and inhibiting the F-2,6-BPase, we would have a relative increase in F-2,6-BP and PFK-2 activity. This would stimulate glycolysis. It makes sense, then, that insulin stimulates PKB.
How is fructose converted into a substrate for glycolysis?
Fructose is phosphorylated into fructose-1-P by fructokinase (sound familiar?). This is cleaved by aldolase B into DHAP (which can be converted into GAP) and glyceraldehyde. Triose kinase then converts glyceraldehyde into GAP.
What enzymes are deficient in essential fructosuria and hereditary fructose intolerance?
Essential fructosuria: Frucotkinase
Hereditary fructose intolerance: Aldolase B
Explain the polyol pathway and the clinical significance of it.
Polyol pathway: Glucose is converted into sorbitol by aldose reductase, which requires NADPH. Sorbitol is converted into fructose by sorbitol dehydrogenase, which produces NADH. However, sorbitol dehydrogenase is not very active, so sorbitol builds up in hyperglycemic states like uncontrolled diabetes. Increased sorbitol levels can lead to cataracts and reduced ability to manage oxidative stress.
When ____ is active, PFK-1 is active!
PFK-2
A laboratory researcher incubated rat hepatocytes in media that contained glucose. She then added forskolin, an activator of adenylate cyclase. How would the concentrations of glucose in the media and fructose-2,6-bisphosphate in the hepatocytes change on addition of forskolin?
Glucose: Increase.
F-2,6,-BP: Decrease.
Forskolin will mimic the effect of glucagon, as glucagon stimulates PKA via the stimulation of adenylate cyclase. This activates gluconeogenesis and glycogenolysis. PFK-2/FBPase-2 is a substrate for PKA. The phosphorylated form of this enzyme has elevated FBPase activity, thus resulting in increased hydrolysis of F26BP. This makes sense, as F26BP would increase glycolysis, which is what we do not want to happen when glucagon is present.
Why are cataracts seen in classical galactosemia?
The excess galactose can travel to tissues such as the lens of the eye. The galactose is converted into galactitol via the polyol pathway (aldose reductase). This galactitol creates an osmolar imbalance in the eye that leads to the development of cataracts.
Symptoms of essential fructosuria?
None!
What are the activators and inhibitors of PFK-1? Explain the role of PFK-2 in PFK-1 metabolism.
Activators: AMP, F-2,6-BP. AMP shows that there is a low energy in the cell and thus we need more glycolysis. F-2,6,-BP shows that there is a high F6P concentration and thus we need more glycolysis.
Inhibitors: Citrate, ATP. Both show that we have high energy in the cell.
If F6P concentrations are high, there is a greater chance that it will be used as a substrate for PFK-2. This will convert it into F-2,6-BP, which is then free to allosterically activate PFK-1, signaling that we need to convert the excess F6P into F-1,6-BP. Hormones can stimulate the activation of PFK-2 to activate PFK-1. F-2,6-BPase will cleave F-2,6-BP when we need to inhibit glycolysis.
What organ does NOT have glucagon receptors??
Skeletal muscle!
A 50-year-old man with a history of type II diabetes presents with complaints of blurred vision and increased sensitivity to light glare, which his physician suspects is due to the development of cataracts. Which of the following is a significant contributor to the development of these symptoms?
A) Increased activity of PFK-2
B) Increased activity of aldose reductase
C) Increased intracellular galactitol concentrations
D) Decreased activity of G6PDH
E) Decreased neuronal ATP levels
The correct answer is B. The development of cataracts in this patient with diabetes is due at least in part to increased activity of aldose reductase, the first enzyme in the polyol pathway.
Which is worse: Essential fructosuria or hereditary fructose intolerance?
Hereditary fructose intolerance. Again, fructoKINase deficiency is KINDer.
A 2-week-old newborn was brought to the pediatrician due to frequent vomiting, lethargy, and diarrhea. Family history revealed that the child never seemed to eat well, and had only been breast-fed. Physical examination revealed an enlarged liver and jaundice. The pediatrician was suspicious of an inborn error of metabolism and referred the child to an ophthalmologist for a slitlamp exam, which revealed cataracts. An enzyme that may be defective in this child is which one of the following?
(A)Fructose-1,6-bisphosphatase
(B) Galactose-1-phosphate uridylyltransferase
(C) Galactokinase
(D) Glycogen synthase
(E) Fructokinase
The answer is B: Galactose-1-phosphate uridylyltransferase. The child has classic galactosemia, a defect in galactose-1-phosphate uridylyltransferase. Due to the accumulation of galactose-1-phosphate, galactokinase is inhibited, and free galactose accumulates within the blood and tissues. The accumulation of galactose in the lens of the eye provides substrate for aldose reductase, converting galactose to its alcohol form (galactitol). The accumulation of galactitol leads to an osmotic imbalance across the lens, leading to cataract formation. Note that there is also hepatomegaly and jaundice present.