Bke2 Biochemistry Exercises

Group exercise: Mechanisms of enzyme regulation

Why is regulation important in biological systems. Discuss!

Answer:
It is probably no exaggeration to say that life on earth is totally dependent on a tight control of all chemical processes in living organisms. This is particularly obvious in complicated organisms like higher plants and mammals, but even a less complicated organism like a bacterium depends entirely on regulation. With its small size and the small size of its genome it is important that no energy and building blocks are wasted in e.g. making metabolites that are not needed. It is left to your imagination to come up with more reasons and answers.
Name as many different modes of regulation you can think of. Which type of modification of the protein is required in each case? Is the modififation reversible or irreversible?

Answer:
For details, see Horton (2nd Ed: Ch 5 pp 137-145 and Ch 6 pp 168-169; 3rd Ed: Ch 5 pp 148-156 and Ch 6 p 186). The main types are:
(1) Allosteric control. Binding (at a regulatory site) of a regulatory molecule in addition to the substrate. Reversible.
(2) Proteolytic activation. Peptide bonds in the protein are cleaved (hydrolysed) by a protease. Irreversible.
(3) Reversible covalent modification, e.g. phosphorylation. Phosphorylation is catalysed by protein kinases, which transfers a phosphate group from ATP to a Ser, Thr or Tyr residue on the enzyme. The reverse, i.e. removal of the phosphate group is performed by protein phosphatases (or protein phosphorylases).
(4) Binding of regulatory protein. Reversible.
(5) Gene regulation. Does not affect the properties of the enzyme, but the amount.
Discuss and try to explain allostery. Allostery can be homotropic or heterotropic. Explain. What does positive cooperativity mean? Which glycolytic enzymes are allosterically regulated, and how?

Answer:
The word allosteric comes from the Greek words meaning "other shape". It means that the protein has (at least) two different shapes (conformations) that have different functional properties. The relative amounts of the two states can be changed by binding things at the active, and other, sites. So, binding at one site can change the properties of another site on the protein. Allosteric interactions which involve two different kinds of sites are termed heterotropic allosteric interactions; they may occur within the same enzyme molecule (e.g. the Bohr effect in hemoglobin).

Allosteric proteins often have more than one subunit, and so they also can generally communicate this kind of information from one active site to that on another subunit (also through conformational changes). Binding of substrate on one subunit affects binding of the same substrate on the other subunits. Such homotropic allosteric interactions give rise to cooperativity in the binding, which is nearly always positive (e.g. cooperative oxygen binding in Hb).

Allosteric effects on activity may be positive or negative. Thus, allosteric regulators can act either as inhibitors or activators.

Several of the enzymes involved in glycolysis are allosterically regulated. The reaction catalysed by hexokinase is metabolically irreversible. Hexokinase is under feedback control of the product of this reaction (glucose-6-phosphate), which allosterically inhibits hexokinase. The form of hexokinase which predominates in the liver (hexokinase IV, glucokinase) is an exception, and can continue to produce glucose-6-phosphate for use in glycogen synthesis independent of the rate of glycolysis in other tissues.

Phosphofructokinase (PFK) also catalyses a metabolically irreversible reaction in glycolysis. This enzyme is allosterically regulated in several ways. AMP and fructose 2,6-bisphosphate activate the enzyme, whereas ATP and citrate inactivate the enzyme. Thus, when energy (ATP) and citric acid cycle intermediates (citrate) are abundant, PFK is inhibited. When energy consumption is high (AMP), or blood glucose is high (fructose 2,6-bisphosphate), PFK is activated.

The third point of control of glycolysis is pyruvate kinase, which again catalyses a metabolically irreversible reaction. This enzyme is activated by fructose 1,6-bisphosphate (the product of the reaction catalysed by PFK, so this is an example of feed forward activation), and inhibited by ATP. (Pyruvate kinase is further controlled through reversible covalent modification: phosphorylation of the enzyme renders it less active).
If you separated hemoglobin into dimers of (a+b) subunits, would you expect the dimers to bind more or less O₂ at low O₂ pressures? Explain. What effect would 2,3-bisphosphoglycerate have on oxygen binding?

Answer:
Cooperativity in the tetrameric hemoglobin depends on the interaction of the two a and the two b subunits. Separation of the tetramer into two a-b dimers would destroy the cooperativity exhibited by the tetramer. There should be more O₂ bound at lower O₂ pressures. The O₂-binding curve for the dimer should resemble that of myoglobin. 2,3-Bisphosphoglycerate should not stabilise the deoxyform of the dimer and would therefore not have any effect on oxygen binding.
What does the curve for the dependence of the reaction velocity on the substrate concentration for an allostrically controlled enzyme look like? Do these enzymes follow Michaelis-Menten kinetics?

Answer:
The curve has sigmoidal shape instead of hyperbolic. Allosterically regulated enzymes do not follow Michaelis-Menten kinetics.
Explain the term feedback control. Why is it necessary?

Answer:
The enzyme that catalyses the first unique step (committed step) in a biosynthetic pathway is often inhibited by the ultimate product. This so called feedback control (feedback inhibition) is one way for the living cell of economising by controlling that just enough, but not too much, product is produced. See Horton, p 303 in 2nd Ed; p 314 in 3rd Ed.
Discuss the details of proteolytic activation. What may be the reasons for using protelytic cleavage as the means to control e.g. blood clotting and digestive enzymes?

Answer:
The enzyme is first synthesised as a precursor protein that is inactive, a zymogen. It is stored and/or transported in its inactive form until it is needed. Then it can be activated by a protease that cleave specific peptide bond(s) in the enzyme. It is vital that these proteins are only active when and where they are required. For example, digestive enzymes are synthesised in the pancreas but active in the gut. Blood clotting proteins are circulating in the blood and shall absolutely not be active except from at the site of an injury. The multistep proteolytic activation of blood clotting give rise to a rapid amplification of the initial signal, also called a cascade effect. See Horton, pp 168-169 in 2nd Ed; p 186 in 3rd Ed.

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