Glycolysis

Question 1

Glycolysis is the oxidation of _____ to ______

Answer 1

glucose (6 carbon molecule); pyruvate (or lactate)

Question 2

Glycolysis occurs where?

Answer 2

cytosol

Question 3

Three Stages of glycolysis

Answer 3

1. Conversion of glucose to fru-1,6-bisphosphate (uses 2 ATPs)
2. Cleavage of fru-1,6-bisphosphate to DHAP and glyceraldehyde-3-phosphate (G3P)
3. Conversion of G3P to pyruvate (produces 4 ATPs and 2 NADHs)

Question 4

Pyruvate has at least two fates:

Answer 4

1. complete oxidation to CO2
2. fermentation to lactate

Question 5

Anaerobic glycolysis is the only energy source in:

Answer 5

• Red blood cells
• Cornea and lens of eye, and certain regions of the retina
• Renal medulla
• Testis
• Leukocytes
• White (fast-twitch) muscle fibers

Question 6

Main fuel for brain

Answer 6

Glucose

Question 7

Glycolysis is an important pathway in tumor cells the ________effect and in embryonic stem cells in which a ________ occurs during implantation

Answer 7

Warburg; “metabolic shift”

Question 8

Step #1:

Irreversible (i.e., equilibrium lies far to right)

In muscle allosterically inhibited by:

Cofactor

Answer 8

Conversion of Glucose to Glucose-6-Phosphate (via Hexokinase)

Inhibited by: Glu-6-P (in muscle)

Irreversible step

Cofactor: Mg²⁺

Question 9

Step #2

Answer 9

Isomerization of Glucose-6-Phosphate to Fructose-6-Phosphate (via Phosphoglucose isomerase)

Reversible

Question 10

Step #3

Cataylst Inhibitors:

Activators:

Cofactor:

Answer 10

Conversion of Fructose-6-Phosphate to Fructose-1,6-Bisphosphate (via PFK-1)

Irreversible step

Inhibitors: ATP (liver & muscle), H⁺ (muscle), citrate (liver)

Activators: AMP (muscle), Fru-2,6-bisP (liver)

Cofactor: Mg²⁺

Question 11

Step #4

Answer 11

Cleavage of Fructose-1,6-Bisphosphate (via Aldolase)

yields Dihydroxyacetone phosphate (DHAP) and Glyceraldehyde 3-phosphate (GAP)

GAP continues on down pathway

Question 12

Step #5

Answer 12

The Triose Phosphate Isomerase (TIM)-Catalyzed Reaction

• Isomerization of ketose into aldose: intramolecular redox reaction
• Involves formation of “enediol” intermediate
• “reversible”

Dihydroxacetone phosphate to glyceraldehyde 3-phosphate

Question 13

Step #6

Answer 13

Converstion of Glyceraldhyde-3-Phosphate to 1,3-Bisphosphoglycerate

• Reaction occurs in two stages:
  
  • Oxidation of –CHO to –CO2- using NAD+
  • Joining of –CO2- and Pi to form an acyl-P product, 1,3-BPG
    
    • Two stages must be coupled: favorable oxidation drives unfavorable formation of acyl-P compound
• Coupling occurs via formation of high-energy thioester intermediate involving active site cysteine (Cys-149)
• Overall, two NADHs produced from two NAD’s

Question 14

Step #7

Cofactor:

Answer 14

The Phosphoglycerate Kinase-Catalyzed Reaction

1,3-Bisphosphoglycerate to 3-Phosphoglycerate

Cofactor: Mg²⁺

Substrate-level phosphorylation occurs

Generates two ATPs

Question 15

Step #8

Answer 15

Phosphoglycerate Mutase

reversible

• Catalyzes intramolecular group transfer
• Employs active site phospho-histidine residue
• Catalytic amounts of 2,3-BPG needed to maintain histidine in phosphorylated state
  
  • 2,3-BPG synthesized from 1,3-BPG by special mutase
  • Levels of 2,3-BPG low in most cells (but high in RBCs

Question 16

Step #9

Answer 16

Enolase

reversible

Dehydration of substrate markedly elevates P-group transfer potential

Question 17

Step #10

Answer 17

Pyruvate Kinase

irreversible

• Energy that’s trapped by P within unstable enol form is released when enol changes to more stable ketone form upon loss of P: enol-ketone conversion drives reaction
• Substrate-level phosphorylation occurs; 2 more ATPs produced

Question 18

Step #11

Answer 18

Regeneration of NAD⁺ under anaerobic conditions

• Lactic acid fermentation; it occurs under O2-limiting conditions
• Is necessary to maintain REDOX balance
• (NADH, NAD+) in limited supply, so regeneration critical
• NAD+ necessary for G3P DHase step; without NAD+, glycolysis would come to a standstill

Question 19

Modes of Regulation of Glycolytic (and other) Enzymes

Answer 19

• Allosteric control
• Covalent modification via protein phosphorylation and dephosphorylation
• Different isozymic forms
• Transcriptional control (enzyme induction)
• Sequestration by binding another protein
• Subcellular translocation

Question 20

How is glycolytic flux regulated?

At the irreversible steps catalyzed by the enzymes:

Answer 20

–hexokinase,

–phosphofructokinase, and

–pyruvate kinase

Question 21

Hexokinase

Muscle:

Liver:

Answer 21

• Muscle: Hexokinases I-III are allosterically inhibited by glucose 6-phosphate
• Liver: Glucokinase (HK IV)
  
  • Not inhibited by glucose-6-phosphate
  • Fructose 6-phosphate “substitutes” for Glu-6-P in inhibiting HK IV
    
    • Fru-6P in equilibrium with Glu-6-P in cell
    • Fru-6P’s binding to a nuclear protein, GK-RP, promotes the binding of HK IV to GK-RP, causing its translocation into the nucleus
    • This removes it from cytosol
    • High levels of glucose reverse the effect of Fru-6-P
  • Insulin indirectly increases HK IV protein level through stimulation of transcription

Question 22

Pyruvate Kinase

Two isozymes:

Answer 22

• Muscle type:
  
  • Allosterically regulated
    
    • inhibited by ATP, which indicates high energy charge, and by alanine (one step away from pyruvate), which indicates an abundance of building blocks
    • Activated by fructose-1,6-bisphosphate
• Liver type:
  
  • Allosterically regulated like M-type
  • Subject to reversible covalent modification (phosphorylation) that is under hormonal control
    
    • When blood glucose levels low, glucagon-triggered cAMP cascade leads to phosphorylation of PK; this inhibits it
  • Protein levels also subject to hormonal control
    
    • Insulin stimulates PK gene expression; glucagon inhibits it

Question 23

Phosphofructokinase-1

Answer 23

• Allosterically controlled
• In muscle, PFK activity regulated by energy charge
  
  • Inhibited by ATP
  • Very sensitive to level of AMP, an activator
  • In cell, AMP, as opposed to ADP, is a good regulator, due to:
    
    • Presence of adenylate kinase (ADP + ADP = ATP + AMP)
    • The fact that ATP › ADP › AMP, so that a small decrease in ATP level results in a large increase in AMP level; AMP is more exquisite “sensor” of energy charge
• In liver, allosterically controlled by ATP and citrate (both inhibitors)
• In liver, activated by fructose 2,6-bisphosphate (Fru-2,6-BP), whose level is under hormonal control
  
  • Increased by insulin, which indirectly causes dephosphorylation and activation of a the kinase activity of a bifunctional enzyme (phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK2/FBPase2)) that makes and degrades Fru-2,6-BP
  • Decreased by glucagon, which indirectly causes phosphorylation (and inactivation) of the kinase domain of the bifunctional enzyme

Question 24

Glucose Transporters

Answer 24

• Twelve GLUTs identified, but only five have known functions
• Each comprises single polypeptide chain, is ≈ 500 amino acids long and has 12 transmembrane segments; forms pore in membrane
• Each is an “enzyme” whose function is to transport glucose
• Km-values differ according to physiological role & tissue location