Chapter 14. Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway

Question 1

Explain Hexokinase

Answer 1

• Large negative ΔG due to ATP coupling, irreversible
• Mannose and fructose also substrates for some subtypes
• Large induced fit occurs with substrate binding
• Hexokinase phosphorylates glucose, helps it stay in cell
• Occurs as dimer; 4 subtypes; liver form: glucokinase

Question 2

Explain Induced Fit of Hexokinase

Answer 2

•Large induced fit occurs with substrate binding

2 lobes close, envelops ATP and glucose, 8oA movement

Active site environment becomes non-polar: facilitates nucleophilic attack: proton on C6 OH of glucose may be removed prior to attack of the terminal phosphorus of ATP

Excludes water and prevents simple ATP hydrolysis (ATPase)

Enzyme catalysis after both glucose, ATP bound, no order

Induced fit common feature of kinases •Inhibited typically by product G6P, PO43- relieves inhib

Question 3

What is the most regulated enzyme?

Answer 3

PFK 1 (phosphofructokinase 1)

Question 4

What activates PFK-1?

Answer 4

• increase in Fructose 2,6 biphosphate (THIS IS NOT same as F-1,6-BP)
• increase in ADP and AMP
• increase in xylulose 5 phosphate

Question 5

What inhibits PFK-1?

Answer 5

• Increase in ATP
• Increase in Citrate

Question 6

How does the increase in glucagon affect PFK1 and, in turn, affect glycolysis and gluconeogenesis?

Answer 6

Increasing glucagon (an inhibitor) decreases Fructose 2,6 BP which decreases PFK-1 which decreases glycolysis and increases gluconeogenesis

Question 7

What are other regulators of PFK1?

Answer 7

• Xylulose 5-phosphate, part of the pentose phosphate pathway, activates phosphoprotein phosphatase 2A (PP2A)
• PP2A dephosphorylates several target proteins, including PFK2/FBPase2
• This shifts balance toward glycolysis, acetyl-CoA production, and stimulates enzymes for lipid synthesis in the liver.
• High phosphoenolpyruvate inhibits PFK1, i.e. if pyruvate kinase is blocked, PFK1 blocked

Question 8

Explain the importance of Fructose-2,6-Bisphosphate.

Answer 8

• Primary reciprocal regulator of glycolysis vs glyconeogenesis
• Potent activator of PFK1/glycolysis and inhibitor of gluconeogenesis (FBPase-1)
• PFK-2 phosphorylates F6P to make F-2,6-bP (promotes glycolysis)
• F-2,6-bP hydrolyzed by FBPase2 (favors gluconeogenesis)
• Both kinase and phosphatase activities on same bi-functional enzyme
• Hormonally regulated

Question 9

Explain Step 5 - Triose Phosphate Isomerase

Answer 9

• Interconverts DHAP and GAP, only GAP is processed further as part of glycolysis
• DHAP slightly favored but converted to GAP because GAP depleted by next step in glycolysis
• Tetramer, active site is an alpha/beta barrel
• Glu 165 initiates reaction as a general base, abstracts proton alpha to carbonyl; if genetically modified to Asp slows reaction 1000 fold by placing the carboxylate group 1oA farther away from DHAP
• Enediol intermediate (symmetric, easily reversible; stable ene-diol transition state analogues bind 100 fold better than substrate; inhibit the reaction (enzyme stabilizes transition state)

Question 10

Triosphosphate Isomerase cont.

Answer 10

• A 10 amino acid flexible loop (flap) at the active site overlays the substrate after substrate binding; stabilizes ene-diol intermediate (7 Ao movement)
• This loop prevents the substrate adopting conformations that lead to spontaneous elimination of the phosphate group
• Elimination of the phosphate during catalysis leads to methyl glyoxal formation
• Methyl glyoxal is a potent cell toxin
• “Kinetically perfect” enzyme, i.e. efficiency > 107

Question 11

Explain pyruvate kinase

Answer 11

• Dimer, mechanism analogous to other kinases
• ATP, alanine, acetyl CoA, fatty acids inhibit •AMP, F-1,6-bP activate (feed-forward act.)
• Different isoforms (Type L and Type M)
• Type L in liver [Low glucose leads to inc. cAMP (glucagon from pancreas) which leads to phosphorylation and inactivation of enzyme]
• Type M in muscle and brain

Question 12

What are the main functions of the Pentose Phosphate Pathway?

Answer 12

1. Generate NADPH for some anabolic pathways
2. Produce ribose, part of many important biomolecules.

Question 13

Why NADPH from Pentose Phosphate Pathway?

Answer 13

• NADPH, phosphate provides unique tag
• NADPH, NADH not usually interchangeable [NADP+/NADPH ~0.014 in well-fed rat liver (NAD+/NADH ~700)]
• Used extensively in fatty acid synthesis
• Path very active in tissues that synthesize fatty acids or compounds requiring NADPH

Question 14

What important molecules does the ribose from the Pentose Phosphate Pathway help create?

Answer 14

RNA, DNA, ATP, NAD+, FAD, Coenzyme A