Exam 2 Lecture #4 (Glycolysis Overview) Flashcards
What reactions do the following enzymes catalyze?
Hexokinase
PFK1
Pyruvate Kinase
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
What are the two reactions in glycolysis that are “subrate level phosphorlyation”
Substrate Level Phosphorylation:
1,3 BPG into 3PG by phosphoglycerate kinase
PEP into pyruvate via pyruvate kinase
What is the purpose of the first step of glycolysis?
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
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)
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
Draw and explain the substrate saturation curves for hexokinase and glucokinase
Remember, hexokinase has a low Km (high affinity, very saturated)
Glucokinase has a high Km (low affinity, not saturated at phsyiological glucose concentrations)
Explain the regulation of glucokinase.
What causes it to translocate?
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
Explain the allosteric regulation of PFK-1
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)
What does PFK2 do?
Explain the regulation of PFK2?
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
What happens to hepatic PFK-2 in the presence of glucagon or epinephrine?
Hepatic PFK-2 is phosphorylated in the kinase domain by PKA in response to glucagon or epi.
THIS INACTIVATES it
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?
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)
_______ activates heart and skeletal muscle PFK-2 by inhibiting phosphatase activity.
EPI (but not glucagon) activates heart and skeletal muscle PFK-2 by inhibiting phosphotase activity.
Hepatic PFK-2 is inhibited by phosphorylation of which domain?
Skeletal and Heart PFK-2 activity is activated by phosphorlyation of which domain?
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
In the heart and muscle _____ activates PFK-2 and glycolysis.
In the heart and muscle, EPINEPHRINE activates PFK-2 and glycolysis.
Explain the regulation of pyruvate kinase
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.
Glucagon and Epinephrine cause hepatic pyruvate kinase to become ________.
Glucagon and epinephrine (low blood glucose levels) cause hepatic pyruvate kinase to become phosphorylated and therefore inactive.
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?
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.
Explain what happens with pyruvate carboxylase genetic deficiency.
Pyruvate Carboxylase Genetic Deficiency:
- leads to increase in blood alanine, pyruvate, and lactate levels
- Diagnosis: reduced head circumference (microcephaly), mental retardation, poor muscle coordination
NAD+ needs to be regenerated to maintain glycolytic flux: explain the ways it can be regenerated.
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+)
What happens to lactate levels in anaerobic conditions?
In anaerobic conditions, lactate levels INCREASE
In skeletal muscle, the LDH5 M4 Isozyme “prefers” to catalyze the conversion of ______ to ______.
What does that do?
In skeletal muscle, the LDH5 isozyme prefers to catalyze the conversion of pyruvate to lactate
This allows for high bursts of energy
In heart muscle, the H4 isozyme prefers to catalyze the conversion of ______ to _______.
In heart muscle, isozyme prefers conversion of lactate to pyruvate. This allows for sustained production of energy
What is the normal serum concentration of lactate to pyruvate?
Normal serum concentration is 10/1 of lactate to pyruvate
Give an overview of the PDH complex.
PDH complex is a multienzyme complex (E1, E2, E3) that catalyzes the conversion of pyruvate and coenzyme A (CoASH) into acetyl coA.
Explain the regulation of PDH?
Effects of Glucagon and Epi?
Effects of end products?
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
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?
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
Explain the effects of Lactate Dehydrogenase A Deficiency.
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
Explain how arsenic posioning is related to LDHA
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
Glycolysis can function in both ____ and ____ conditions.
Net ATP production of glycolysis is ___.
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)
Explain what glucose can get turned into with no oxygen.
What processes require O2?
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.
Anaerobic conditions favor the formation of ____
Oxygen is required for the following processes:
Reoxidation of mitochondrial NADH, formed from__
Reoxidation of cytosolic NADH by _______
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
Low levels of cellular NADH cause what?
Low levels of cellular NADH decrease lactate formation
What are the two key enzymes in the metabolism of galactose?
Enzymes in the metabolism of galactose:
- Galactokinase
- Gal-1-P uridlytransferase
What is galactosemia?
Which version is worse?
What happens to patients that have it?
How can it be treated?
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)
Explain what happens in Aldolase B deficiency
What are the symptoms?
What is the treatment?
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
Regulation of Pyruvate Kinase:
What molecules positively regulate pyruvate kinase?
Which molecules negatively regulate pyruvate kinse?
Why?
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
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?
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
In terms of ATP production, how many molecules of ATP per glucose due you get in aerobic vs anaerobic conditions.
Anaerobic: 2 net ATP per glucose
Aerobic: 32 ATP per glucose
In terms of lactate dehydrogenase isozymes, what indicates a heart attack/myocardial infarction
If LDH-1> LDH-2
(meaning, if in the blood there are more heart versions than circulatory version)
