Major concepts:

1. Which glycolysis reactions require different enzymes for gluconeogenesis? Why?
2. How is skeletal myocyte (muscle cell) hexokinase regulation different from hepatocyte (liver cell) hexokinase regulation? How is regulation in the two cells similar?
3. What is the function of fructose-2,6-bis-phosphate, and how is its concentration controlled?
4. What is the effect on the regulated enzymes of glycolysis of increasing the concentration of
(a) ATP, (b) AMP, (c) glucose or glucose-6-phosphate, (d) acetyl-CoA (AcCoA)
What does each of these modulators indicate about energy conditions in the cell?
5. How do insulin, epinephrine, and glucagon affect glycolysis and gluconeogenesis?

Core knowledge:

1. What are isozymes, and how do the isozymes of hexokinase differ from each other?
2. How are PFK-1 and FBPase-1 regulated? What conditions promote PFK-1 activity?
3. How are PFK-2 and FBPase-2 regulated? Why is their regulation significant?
4. How is myocyte pyruvate kinase activated or inhibited?
5. How is hepatocyte pyruvate kinase activated or inhibited?
6. What regulated enzymes are affected by insulin, and what are the effects?
7. What regulated enzymes are affected by glucagon, and what are the effects?
8. What regulated enzymes are affected by epinephrine, and what are the effects?

Methods of enzyme regulation (= metabolic pathway regulation)                                    Fig. 15-2
A. Regulation of enzyme synthesis and degradation – not covered in 340
B. Sequestering enzymes inside organelles = localizing pathways within the cell
C. Changing [substrate]
D. Allosteric regulation = response to changing [modulator]
E. Covalent regulation = response to kinase/phosphatase, which are both regulated
F. Binding a regulatory protein = variation on allosteric regulation

Key points overall in metabolic regulation:
A. The goals of metabolic regulation are
     1. maximize efficient use of fuel (prevent simultaneous operation of pathways that
              reverse each other, such as glycolysis and gluconeogenesis)
     2. allocate fuel resources appropriately for pathways that branch from one metabolite
     3. use the most appropriate fuel (glucose, fatty acid, glycogen, amino acids)
     4. respond appropriately to changes in [metabolite]       (5^th edition, p. 577)
B. The most useful allosteric modulators are those whose concentration varies significantly.
     ATP has a small relative change in concentration, normally, but AMP can have a very
     large relative change when [ADP] increases, as a result of adenylate kinase activity.

Big picture: Pathways of glycolysis and gluconeogenesis +
     Fate of pyruvate
         → oxaloacetate (pyruvate carboxylase) → PEP (PEP carboxykinase) in gluconogenesis
         ↔ lactate (lactate dehydrogenase) when [NAD⁺] is low
         → AcCoA (pyruvate dehydrogenase) → citrate when [NAD⁺] is high   or
          → AcCoA ↔ fatty acids when energy supplies are high
     Glycolysis: irreversible reactions are catalyzed by hexokinase, PFK-1, pyruvate kinase
     Gluconeogenesis: irreversible reactions are catalyzed by glucose-6-phosphatase,
         FBPase-1, pyruvate carboxylase, PEP carboxykinase

Hexokinase and glucose-6-phosphatase regulation

   A. Major regulation = hexokinase isozymes:
      versions present in different cells with different K_M's, different allosteric responses
     1. Myocyte (skeletal muscle):
          hexokinase II has K_M ∼ 0.1 mM, allosteric inhibition by glucose-6-phosphate
     2. Hepatocyte (liver) has hexokinase I-III (function at low [glucose]) and
         hexokinase IV (glucokinase) has K_M ∼ 10 mM,
               allosteric inhibition by a regulator protein that sequesters glucokinase (nucleus)
                  in response to high fructose-6-phosphate concentration
              relief of inhibition when glucose concentration increases
B. Glucose-6-phosphatase regulation: none (enzyme is only present in liver cells)

PFK-1 and FBPase-1 regulation

    A. Allosteric regulation of PFK-1
     1. Activation by AMP, ADP, and fructose-2,6-bis-phosphate
     2. Inhibition by ATP and citrate
B. Allosteric regulation of FBPase-1 = Inhibition by fructose-2,6-bis-phosphate
C. Role of fructose-2,6-bis-phosphate is only regulation; not a metabolic intermediate
     1. Synthesis is by PFK-2: ATP + fructose-6-P → ADP + fructose-2,6-bis-P
     2. Degradation is by FBPase-2: fructose-2,6-bis-P + H₂O → fructose-6-P + P_i

   D. Regulation of PFK-2 and FBPase-2
     1. These are one protein with two activities.
     2. Kinase active site transfers P from ATP to C2 of fructose-6-P
     3. Hydrolase active site adds H₂O to remove P from C2 of fructose-2,6-bis-phosphate.
     4. Only one site is active at a time:
         a. Kinase site is active when the protein is not phosphorylated.
         b. Hydrolase site is active when the protein is phosphorylated.
     5. Glucagon and epinephrine cause phosphorylation by increasing [c-AMP].
     6. Insulin causes removal of phosphate by activating phosphoprotein phosphatase.

Pyruvate kinase and pyruvate carboxylase regulation
A. Isozymes of pyruvate kinase
     1. Muscle pyruvate kinase has allosteric regulation only
     2. Liver pyruvate kinase has allosteric + covalent regulation
B. Allosteric regulation of pyruvate kinase:
     1. allosteric activation by fructose,1-6-bis-phosphate
      2. allosteric inhibition by ATP, AcCoA, fatty acids, alanine
C. Covalent regulation of pyruvate kinase
     1. phosphorylation by PKA = inactivation
     2. removal of phosphate by protein phosphatase = activation
D. Allosteric regulation of pyruvate carboxylase = activation by AcCoA

Carbohydrate metabolism is related to fat metabolism and to protein metabolism.

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