CHEM 440
Biochemistry I
J. D. Cronk   Syllabus [ Previous | Next ] Pick a lecture:
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Lecture 33. Electron transport chain

Monday 7 December 2009

The integral membrane complexes of the mitochondrial ETC.

Reading: BTS6 - Ch.18, pp.509-520.
33. Summary

Lecture 33 Summary

<head>(M 12/3) The integral membrane complexes of the mitochondrial ETC. (VVP3e: Ch.18, pp.602-618.)</head>

The integral membrane complexes of the mitochondrial electron transport chain (ETC).

Gluconeogenesis utilizes some of the same reactions and enzymes of glycolysis, it must circumvent three essentially irreversible steps of glycolysis by alternative reactions that are more energetically favorable. The glycolytic reaction catalyzed by pyruvate kinase, the final such irreversible step, is bypassed in gluconeogenesis by a two-step process. First, pyruvate must be carboxylated to form oxaloacetate, a reaction that is driven at the expense of a high-energy phosphate bond of ATP and is catalyzed by the enzyme pyruvate carboxylase. The next step - conversion of oxaloacetate to phosphoenolpyruvate (PEP) - is also an energy-requiring one, this time utilizing the high-energy phosphate bond of GTP and catalyzed by phosphenolpyruvate carboxykinase. The net reaction for the combination of the two steps is:

pyruvate + ATP + GTP + H2O--> PEP + ADP + GDP + Pi + 2 H+
This reaction has a ΔG°′ (biochemical standard state free energy change) of +0.2 kcal/mol. The next reaction specific to gluconeogenesis is the hydrolysis of fructose1,6-bisphosphate to fructose 6-phosphate catalyzed by fructose 1,6-bisphosphatase [EC 3.1.3.11]. Finally, another dephosphorylation reaction converts glucose 6-phosphate to glucose. This hydrolytic reaction is catalyzed by glucose 6-phosphatase [EC 3.1.3.9]. In human biochemistry, glucose 6-phosphatase is mainly an enzyme of the liver, involved in the regulation of blood glucose levels.

Mechanisms of gluconeogenic enzymes

Pyruvate carboxylase -

Pyruvate carboxylase [EC 6.4.1.1] is a mitochondrial enzyme that is a homotetramer of 120kD subunits, each carrying a covalently-linked biotin prosthetic group. Pyruvate carboxylase is a member of a group of biotin-dependent carboxylase enzymes, which also includes acetyl CoA carboxylase [EC 6.4.1.2] and propionyl CoA carboxylase [EC 6.4.1.3]. The latter two enzymes play important roles in fatty acid metabolism. All the biotin-dependent carboxylase reactions require ATP hydrolysis as well as participation of biotin prosthetic group.

Phosphoenolpyruvate carboxykinase (PEPCK)

Oxaloacetate is diverted to gluconeogenesis by PEPCK [EC 4.1.1.32] in a reaction that uses GTP as the donor of the phosphate that ends up in PEP, and eliminates the CO2 that was incorporated . The PEPCK reaction is therefore the decarboxylation of a β-keto acid coupled to a phosphoryl transfer to the enolate oxygen that would be the expected intermediate of this type of decarboxylation. Energetically, the favorable decarboxylation helps drive the formation of the enol phosphate, which has a significantly higher standard free energy of hydrolysis than the phosphoanhydride bonds of ATP or GTP. Furthermore, we see that the conversion of pyruvate to PEP consumes two high energy phosphate bonds.

PEPCK is a 74 kD monomeric enzyme whose subcellular localization varies with species. In some species, it is mitochondrial, and in others it is cytosolic, while in still others (notably including humans) it is roughly equally distributed in both locations. This pattern bears on the transport of metabolites across the inner mitochondrial membrane that is required for gluconeogenesis (see §16.3 in BTS).

Regulation of gluconeogenesis

Reciprocal regulation of glycolysis and gluconeogenesis
Review of allosteric regulation of PFK
Regulation vs. control of metabolism: Steady-state (homeostasis) vs. metabolic flux

Malate shuttle and compartmental cooperation

Oxaloacetate produced in the mitochondrial matrix by pyruvate carboxylase has two possible fates. Either it is produced for an anaplerotic purpose, in which case it is combined with acetyl CoA to produce citrate for the citric acid cycle, or the oxaloacetate is reduced back to malate, which is then exported to the cytosol where it contributes to gluconeogenesis.

Substrate cycles

If the rates of two directly opposing reactions, the phosphofructokinase reaction of glycolysis and the fructose 1,6-bisphosphatase are measured (with the help, say, of isotope-labeled substrates) it is found that both reactions can and do take place at the same time. Since the net result of this substrate cycle is simply the hydrolysis of ATP, it was believed at one time that this was a wasteful "futile cycle". Subsequently, it was discovered that substrate cycles can provide an effective means to create large changes in metabolic flux.
Diagram of substrate cycle
 

Learning objectives

  • Describe the gluconeogenic pathway: Inputs and outputs, net reaction, names of intermediates, unique steps
  • Recognize the structures of all intermediates in glycolysis and gluconeogenesis
  • Describe the energetics and mechanism of biotin-dependent carboxylases, such as pyruvate kinase.
  • Explain the meaning and metabolic logic of reciprocal regulation of glycolysis and gluconeogenesis.
  • Describe how regulation of substrate cycles contributes to control of metabolic flux
  • Describe the biological role of substrate cycles in thermogenesis.

Page updated 12-17-06

References

  1. Staunton J. Primary Metabolism: A Mechanistic Approoach (1978, Oxford University Press)
  2. Knowles JR. (1989) The mechanism of biotin-dependent enzymes. Ann Rev Biochem 58: 195-221.
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[ E-mail: cronk@gonzaga.edu ]