Question

You have been chosen for a very competitive undergraduate clinical internship at St. Visintainer’s Hospital and Metabolic Clinic working with Dr. Saccharo, an expert in enzyme deficiency disorders related to glycolysis. Before your first day Dr. Saccharo has asked that you review the reactions of glycolysis and the concept of isoenzymes.

Recall that isoenzymes are groups of enzymes that catalyze the same reaction in that they use the same reactants to product the same products. They may have different genes, tissue expression, developmental timing of expression, and affinities for their substrates. As an example, phosphofructokinase-1 has 3 isoenzyme forms: PFK-L is expressed in the liver and the gene is found on chromosome 21; PFK-M is expressed in the muscle and the gene is found on chromosome 12; and PFK-P is expressed in platelets and the gene is found on chromosome 10.

Question 1:

If a person would be deficient in all 3 forms of PFK-1, this person would                  .

A.           Have lower than normal glycogen stores.

B.           Have more mitochondrial activity than normal.

C.           Be dead.

D.          Produce more energy per glucose molecule because glycolysis would be unregulated.

It is now Monday morning and you are ready to start seeing patients with Dr. Saccharo.

Patient 1:

Ann is a teenager and avid golfer who was referred to the clinic after being refused at the blood drive and tiring easily on the high school golf course during practice. Ann is examined and a blood sample was obtained for analysis of a glycolysis related metabolic panel. The results of her panel is below (levels: + = normal, - = decreased; ++= increased):

Blood Metabolic Panel
Blood Glucose	+
Glucose-6-phosphate	++
Fructose-6-phosphate	++
Fructose-1,6,bisphosphate	++
Glyceraldehyde-3-phosphate	++
1,3-bisphosphoglycerate	++
Phosphoenolpyruvate	++
Pyruvate	-
ATP	-
Red Blood Cell Concentration	-

Question 2:

Assuming that Dr. Saccharo is correctly assuming that Ann has a defect in glycolysis, what glycolytic enzyme is the most likely to have the defect based on the blood metabolic profile?

A.           Hexokinase

B.           Phosphofructokinase

C.           Triose Phosphate Isomerase

D.          Pyruvate Kinase

E.           Aldolase

Patient 2:

Marie is a 32-year-old mother of three complaining of fatigue and muscle cramps with exercise. She had always blamed her intolerance to exercise on her sedentary lifestyle. However, she recently joined a gym and after a week of aerobics classes went to her physician, who ordered a series of blood tests. The blood work came back with abnormal results, leading to her muscle biopsy and referral to the metabolic clinic. Marie’s blood was subjected to the same metabolic panel used before with the following results:

Blood Metabolic Panel
Blood Glucose	+
Glucose-6-phosphate	++
Fructose-6-phosphate	++
Fructose-1,6,bisphosphate	-
Glyceraldehyde-3-phosphate	-
1,3-bisphosphoglycerate	-
Pyruvate	-
ATP	-
Red Blood Cell Concentration	-

Marie’s muscle biopsy was also analyzed and showed an excess of glycogen.

Question 3:

What is the most likely enzyme deficiency for Marie?

A.           Hexokinase

B.           Phosphofructokinase

C.           Triose Phosphate Isomerase

D.          Pyruvate Kinase

E.           Aldolase

Question 4:

If you wanted to test the ability of red blood cells to complete glycolysis what compound would you try to detect?

A.           Fructose-6-phosphate

B.           Aldolase

C.           Pyruvate

D.          Phosphofructokinase

E.           Dihydroxyacetone phosphate

Question 5:

If you used a sample of Marie’s blood for this, what compound could you add to enable these cells to complete glycolysis?

A.           Glucose

B.           Fructose-1,6-bisphosphate

C.           Insulin

D.          Glucagon

E.           Fructose-6-phosphate

Answer 1

Answer 1: Isoenzymes are group of enzymes that catalyze the same reaction in that they use the same reactants to product the same products. One such example is of phosphofructokinase-1 which has three isoenzyme forms- PFK-L is expressed in the liver and the gene is found on chromosome 21; PFK-M is expressed in the muscle and the gene is found on chromosome 12; and PFK-P is expressed in platelets and the gene is found on chromosome 10. PFK is the most important rate limiting enzyme for glycolysis pathway. PFK utilizes glucose to produce pyruvate which finally produces ATP via electron transport system. But the deficiency of PFK-1 interferes with the ability of the muscles to use glucose for energy. If a person lack all the three forms of PFK-1, then he will be dead. This is because PFK-1 deficiency results in accumulation of glycogen in the tissue. It can also lead to increased uric acid production with the compensation of haemolytic anaemia. In muscle tissue, PFK-1 is composed of only PFK-M while in erythrocytes, it is composed of both PFK-L and PFK-P. So all forms of PFK-1 deficiency results a person to be dead. Option (C) is the correct answer.

Answer 2: There is an increased level of Glucose-6-phosphate, Fructose-6-phosphate. Fructose-1,6-biphosphate, Glyceraldehyde-3-phosphate, 1,3-biphosphoglycerate and phosphoenol pyruvate while a decreased level of pyruvate, ATP and RBC concentration. The resulting enzyme deficient in Ann is Pyruvate Kinase. Pyruvate kinase catalyzes an irreversible reaction step and is a regulatory enzyme of glycolysis where 1 mole of ATP is generated via substrate level phosphorylation. As there is a high concentration of G6P, F6P, etc it inactivtes pyruvate kinase by phosphorylating it. Option (D) is the correct answer.

Answer 3: Marie's muscle biopsy showed an excess of glycogen with decreased level of Fructose-1,6-biphosphate, Glyceraldehyde-3-phosphate, 1,3-biphosphoglycerate, pyruvate, ATP and RBC. So here the most likely enzyme deficient in Marie is Phosphofructokinase. PFK catalyze the reaction between Fructose-6-phosphate to fructose-1,6-biphosphate. PFK is the most important rate limiting enzyme of glycolysis which catalyzes the second phosphorylation step of glycolysis using a molecule of ATP. It is an allosteric, inducible, regulatory enzyme. Its deficiency result in accumulation of glycogen in the body where glucose is not utilized resulting in excercise intolerance, muscle cramping, exertional myopathy, compensated haemolysis and myoglobinuria. Option (B) is the correct answer.

Answer 4: RBCs do not have mitochondria, so produce energy carrier ATP by glycolysis of glucose and lactic acid fermentation on the resulting pyruvate. So if we want to test the ability of RBCs to complete glycolysis, we should detect the presence of pyruvate, as pyruvate is the final end product of glycolysis formed from one molecule of glucose. Option (C) is the correct answer.

Answer 5: The compound that needs to be added to enable the RBCs to complete glycolysis is Fructose-1,6-biphosphate. This is because Fructose-1,6-phosphate increases the activity of phosphofructokinase (PFK) which is the most important rate limiting enzyme for glycolysis. F-1,6-BP is formed from fructose-6-phosphate by the action of an enzyme called PFK-2. When glucose supply is in plenty, PFK-2 is dephosphorylated and activated. So, F-1,6-BP concentration increases which in turn activates PFK. Thus glycolysis is favored. Option (B) is correct answer.