Exam 2 Lecture #4 (Glycolysis Overview)

Question 1

What reactions do the following enzymes catalyze?

Hexokinase

PFK1

Pyruvate Kinase

Answer 1

Hexokinase: uses ATP to phosphorylate glucose into G6P

PFK-1: uses ATP to turn fructose 6 phosphate into fructose 1, 6 biphosphate

Pyruvate Kinase: (substrate-level phosphorylation)

turns PEP (“phosphoenol pyruvate”) into pyruvate, end up getting ATP production

Question 2

What are the two reactions in glycolysis that are “subrate level phosphorlyation”

Answer 2

Substrate Level Phosphorylation:

1,3 BPG into 3PG by phosphoglycerate kinase

PEP into pyruvate via pyruvate kinase

Question 3

What is the purpose of the first step of glycolysis?

Answer 3

Hexokinase uses ATP to phosphorlyate glucose and create glucose 6-phosphate.

This is important because it creates a polar molecule, and that polar molecule becomes impermeable/ can’t leave the cell

Question 4

Explain the differences between hexokinase and glucokinase.

(where are they present, what is their regulation mechanism, are they inducible or not, what is the Km)

Answer 4

Hexokinase:

• Present in all cell types
• Allosterically inhibited by its product (negative feedback inhibition)
• Constitutive enzyme, non inducible, constant amount
• LOW Km

Glucokinase:

• present in liver and pancreas
• translocation between nucleus and cytosol (active)
• product decreases activity by promoting translocation to nucleus
• INDUCIBLE enzyme: enzyme synthesis increased by insulin
• HIGH Km

Question 5

Draw and explain the substrate saturation curves for hexokinase and glucokinase

Answer 5

Remember, hexokinase has a low Km (high affinity, very saturated)

Glucokinase has a high Km (low affinity, not saturated at phsyiological glucose concentrations)

Question 6

Explain the regulation of glucokinase.

What causes it to translocate?

Answer 6

High glucose and fructose cause GK to translocate to the cytosol where it is active

High fructose 6-P causes GK to translocate to the nucleus where it is inactive.

Note: fructorse 1 phosphate promotes the activity of GK by inhibiting it from moving back to nucleus

Question 7

Explain the allosteric regulation of PFK-1

Answer 7

Inactivation: Higher levels of citrate and ATP inhibit PFK-1

Activation: Higher leves of fructose 2, 6 bisphosphate, AMP, and ADP positively activate PFK-1

(activity of PFK-1 is regulated by the product of PFK-2 aka fructose 2, 6 P)

Question 8

What does PFK2 do?

Explain the regulation of PFK2?

Answer 8

PFK-2 takes fructose 6-P and uses ATP to turn it into fructose 2, 6, P (which activates PFK1)

High blood glucose and high insulin upregulates this process

Low blood glucose and high glucagon/epi downregulates this process

Question 9

What happens to hepatic PFK-2 in the presence of glucagon or epinephrine?

Answer 9

Hepatic PFK-2 is phosphorylated in the kinase domain by PKA in response to glucagon or epi.

THIS INACTIVATES it

Question 10

What happens to hepatic PFK-2 in presence of glucagon and epi?

What happens to PFK-2 in heart and skeletal muscle in the presence of epinephrine?

Answer 10

In presence of glucagon and EPI:

Hepatic PFK-2 is inhibited via phosphorylation of the kinase domain (glycolysis is inhibited because there is no formation of fructose 2, 6 bisphosphate)

Heart and Skeletal PFK-2: epinephrine increases PFK-2 activity by phosphorlyating the phosphotase domain (inhibiting the phosphotase activity, and increasing PFK-2 activity)

Question 11

_______ activates heart and skeletal muscle PFK-2 by inhibiting phosphatase activity.

Answer 11

EPI (but not glucagon) activates heart and skeletal muscle PFK-2 by inhibiting phosphotase activity.

Question 12

Hepatic PFK-2 is inhibited by phosphorylation of which domain?

Skeletal and Heart PFK-2 activity is activated by phosphorlyation of which domain?

Answer 12

Hepatic PFK-2 is inhibited by Phosphorylation of kinase domain (in presence of glucagon and epi)

Skeletal and Heart PFK-2 activity is ACTIVATED in presence of epi because the phosphotase domain gets phosphorylated

Question 13

In the heart and muscle _____ activates PFK-2 and glycolysis.

Answer 13

In the heart and muscle, EPINEPHRINE activates PFK-2 and glycolysis.

Question 14

Explain the regulation of pyruvate kinase

Answer 14

Pyruvate kinase is regulated by a phosphorylation event (covalent modification).

If there is low blood glucose, and high glucagon and epinephrine, protein kinase A will phosphorlyate pyruvate kinase and make it more inactive.

If there is high blood glucose and high insulin levels, phosphoprotein phosphotase will dephosphorlyate pyruvate kinase and make it active again.

Question 15

Glucagon and Epinephrine cause hepatic pyruvate kinase to become ________.

Answer 15

Glucagon and epinephrine (low blood glucose levels) cause hepatic pyruvate kinase to become phosphorylated and therefore inactive.

Question 16

What do glucagon and EPI do to hepatic glycolysis?

Which two key enzymes do they affect via covalent modification?
Which enzymes do they repress the synthesis of?

Answer 16

Glucagon and Epi inhibit hepatic glycolysis

They do this via inhibiting enzyme activity and repression of mRNA synthesis.

Glucagon and epi cause covalent modification of two key enzymes: inhibiting PFK-2 (decreasing the activity of PFK-1) and inhibiting pyruvate kinase.

Glucagon and EPI repress synthesis of the following enzymes in the liver: glucokinase, PFK-1, and pyruvate kinase.

Question 17

Explain what happens with pyruvate carboxylase genetic deficiency.

Answer 17

Pyruvate Carboxylase Genetic Deficiency:

• leads to increase in blood alanine, pyruvate, and lactate levels
• Diagnosis: reduced head circumference (microcephaly), mental retardation, poor muscle coordination

Question 18

NAD+ needs to be regenerated to maintain glycolytic flux: explain the ways it can be regenerated.

Answer 18

NAD+ can be regenerated by:

• aerobic conditions (oxygen required): NAD+ regenerated by ETC and oxidative phosphorlyation and then uses a metabolite shuttle system
• Anareobic conditions: replenishes NAD via reduction reaction using lactate dehydrogenase (pyruvate is reduced to lactate and NADH is oxidized to NAD+)

Question 19

What happens to lactate levels in anaerobic conditions?

Answer 19

In anaerobic conditions, lactate levels INCREASE

Question 20

In skeletal muscle, the LDH5 M4 Isozyme “prefers” to catalyze the conversion of ______ to ______.

What does that do?

Answer 20

In skeletal muscle, the LDH5 isozyme prefers to catalyze the conversion of pyruvate to lactate

This allows for high bursts of energy

Question 21

In heart muscle, the H4 isozyme prefers to catalyze the conversion of ______ to _______.

Answer 21

In heart muscle, isozyme prefers conversion of lactate to pyruvate. This allows for sustained production of energy

Question 22

What is the normal serum concentration of lactate to pyruvate?

Answer 22

Normal serum concentration is 10/1 of lactate to pyruvate

Question 23

Give an overview of the PDH complex.

Answer 23

PDH complex is a multienzyme complex (E1, E2, E3) that catalyzes the conversion of pyruvate and coenzyme A (CoASH) into acetyl coA.

Question 24

Explain the regulation of PDH?

Effects of Glucagon and Epi?
Effects of end products?

Answer 24

Regulation of PDH:
NOT regulated by Glucagon and Epi

End products inhibit PDH via allosteric inhibition

END products (NADH and acetyl coA) cause phosphorlyation and inhibition of PDH

The end products promote phosphorylization of the pyruvate dehydrogenase (making it inactive) AND allosterically inhibit the enzyme

Question 25

Name the three enzymes in the PDH and name the vitamins they are associated with.

What happens when you have a deficiency in one of those enzymes?

Answer 25

E1: pyruvate dehydrogenase, vitamin is Thiamine/B1

E2: Dihydrolipoyl Transacetylase, vitamin is panto B5

E3: dihydrolipoyl dehydrogenase, (vitamins are riboflavin B2, and niacin B3)

Vitamin Deficiency of B1, B2, B3 or B5 makes PDH less active

Question 26

Explain the effects of Lactate Dehydrogenase A Deficiency.

Answer 26

LDHA patients can’t maintain a moderate level of exercise due to an inability to utilize glycolysis to produce ATP needed under anerobic conditions. (pyruvate can’t be turned into lactate)

When LD levels are insufficient, level of NAd+ becomes limited

Question 27

Explain how arsenic posioning is related to LDHA

Answer 27

arsenic poisoning inhibits the shuttling of the lipoic acid in the oxidized and reduced form, symptoms mimic those of LDHA. Pyruvate and lactate accumulate in the blood and cause lactic acidosis

Question 28

Glycolysis can function in both ____ and ____ conditions.

Net ATP production of glycolysis is ___.

Answer 28

Glycolysis can function in both aerobic and anaerobic conditions.

NET ATP production is 2. (so in anaerobic conditions, the net ATP production is two per glucose)

Question 29

Explain what glucose can get turned into with no oxygen.

What processes require O2?

Answer 29

Glucose can get convereted to pyruvate, and pyruvate can get turned into lactate in ANAEROBIC conditions

There is an O2 requirement for the PDH complex AND for the TCA cycle.

Question 30

Anaerobic conditions favor the formation of ____

Oxygen is required for the following processes:

Reoxidation of mitochondrial NADH, formed from__

Reoxidation of cytosolic NADH by _______

Answer 30

Anaerobic conditions favor the formation of LACTATE

Oxygen is required for:

Reoxidation of NADH in mitochondria from PDH complex

Reoxidation of NADH by mitochondrial linked shuttles

Question 31

Low levels of cellular NADH cause what?

Answer 31

Low levels of cellular NADH decrease lactate formation

Question 32

What are the two key enzymes in the metabolism of galactose?

Answer 32

Enzymes in the metabolism of galactose:

1. Galactokinase
2. Gal-1-P uridlytransferase

Question 33

What is galactosemia?

Which version is worse?

What happens to patients that have it?

How can it be treated?

Answer 33

Galactosemia is a genetic disorder caused by deficiency of either galactokinase or the uridyltransferase….which causes an accumulation of galactitol

Most common and severe form is deficiency in uridyltransferase

Symptoms: cataracts, brain damage, kidney damage

Treatment: remove galactose from diet (lactose)

Question 34

Explain what happens in Aldolase B deficiency

What are the symptoms?

What is the treatment?

Answer 34

Aldolase B Deficiency: can’t cleave fructose 1-P, leading to intracellular trapping of fructose 1-P.

This depletes Pi and ATP levels.

Cells are then damaged because they can’t maintain ion gradients.

Symptoms: hypoglycemia, vomiting, jaundice, liver failure

Treament: avoid foods high in fructose

Question 35

Regulation of Pyruvate Kinase:

What molecules positively regulate pyruvate kinase?

Which molecules negatively regulate pyruvate kinse?

Why?

Answer 35

Regulation of Pyruvate Kinase:

Positive Regulators: Fructose 1,6 bisphosphate

Negative Regulators: ATP, and Alanine

Alanine levels are high during fasted state

Alanine is a significant precursor of gluconeogenesis

Question 36

Explain the regulation on the synthesis of the three irreversible enzymes of glycolysis?

What conditions favor a DECREASE in the synthesis of those three irreversible enzymes?

What conditions favor an INCREASE in the synthesis of those enzymes?

Answer 36

Increased glucagon and epinephrine cause a DECREASE in the synthesis of those three enzymes

Increased insulin, decreased cAMP levels (decreased glucagon and epi) result in increase in synthesis of those enzymes

Question 37

In terms of ATP production, how many molecules of ATP per glucose due you get in aerobic vs anaerobic conditions.

Answer 37

Anaerobic: 2 net ATP per glucose

Aerobic: 32 ATP per glucose

Question 38

In terms of lactate dehydrogenase isozymes, what indicates a heart attack/myocardial infarction

Answer 38

If LDH-1> LDH-2

(meaning, if in the blood there are more heart versions than circulatory version)