BC10M: Introductory Biochemistry, 2006 Semester 2.

1. BC10M: Introductory Biochemistry, 2006 Semester 2. • Thursday 16 Mar. • Lecture 22 • Metabolism & its regulation • Andrew Pearson

2. Metabolism: the sum total of chemical (and physical) changes that occur in living organisms, and which are fundamental to life. Concise Encyclopedia of Biochemistry

3. The metabolic activity represented in this diagram must be controlled for the cell/ organism to survive and reproduce. From Brock

4. Metabolism. All chemical & physical changes that occur in living organisms appear to obey the “universal laws” of thermodynamics. Reactions must be thermodynamically possible, even if seemingly unfavorable, for them to occur in biochemistry.

5. Let us take the case of the glycolytic pathway, which has several enzyme-catalysed steps: From Stryer

6. From Matthews & van Holde.

7. As can be seen, some reactions of glycolysis require an input of energy, whereas others release it. Thus by coupling unfavorable reactions to reactions that can go spontaneously, desired biomolecules can be synthesised without flouting the laws of thermodynamics. Since many of the effective units of metabolism are the metabolic pathways, how are they controlled? Let us go back to look at a simple reaction catalysed by a Michaelis-Menten enzyme.

8. Michaelis-Menten Plot Vmax.[S] KM + [S] Vo =

9. Vmax is the maximum velocity which the amount of enzyme used here can achieve. Vmax.[S] KM + [S] [S] is the concentration of substrate being varied here. Vo = KM is defined as the ratio of the rate constants of the component reactions in the derivation of the rate equation: Enzyme + Substrate  ES complex Enzyme + Product k-1 k2 E + S  ES  E + P k1 k-1+ k2 KM = k1

10. Vmax.[S] KM + [S] Vo = The value of the KM of an enzyme for one of its substrates can be determined as shown on this graph. The value of the KM is not the same as the definition of the KM

11. Vmax.[S] KM + [S] Vo = When the maximum reaction rate/velocity (Vmax) has been reached, increasing the substrate concentration will not increase the reaction velocity. This is because every molecule of enzyme is engaged in a catalytic cycle all the time, and the system cannot be driven faster (without increasing the temperature of the system). The enzyme is said to be saturated with substrate.

12. Vmax.[S] KM + [S] Vo = The majority of enzymes appear to behave according to the Michaelis-Menten model: they sit around waiting for substrate molecules to appear and convert them to product at a rate given by the equation. As they convert the substrate, the [S] decreases and the enzymic conversion rate decreases in compensation.

13. But it is difficult to plot experimental data on a rectangular hyperbola: better to convert the equation to that of a straight line. Vmax.[S] KM + [S] Vo =

14. 1 Vo Km Vmax 1 [S] 1 Vmax = x +

15. Apart from being easily used for the experimental determination of the value of KM, the “double reciprocal” plot can be used to diagnose types of reversible inhibitor, such as competitive and non-competitive. Look these up in a textbook.

16. Regulation of enzyme catalysed reactions.

17. Regulation of enzyme catalysed reactions. Michaelis-Menten enzymes respond only to the concentration of substrate. The only other way a cell can control such a reaction is to make more or less enzyme molecules. This would mean controlling gene expression, and it does occur for some entire metabolic pathways. There are very few circumstances in which either pH or temperature are used to control reactions. Metabolism is routinely controlled by “regulatory enzymes”, which are either allosterically or covalently modulated.

18. Regulation of enzyme activity The main reason for metabolic regulation is the efficient (economic) utilisation of scarce resources.

19. Let us suppose that our simple reaction is A à B

20. Let us further suppose that our simple reaction is part of a metabolic pathway, such as glycolysis, with the following structure: A à B à C à D à E

21. Let us suppose that our simple reaction is part of a metabolic pathway with the following structure; each reaction being catalysed by a separate enzyme: A à B à C à D à E a b c d

22. Let us suppose that our simple reaction is part of a metabolic pathway: each reaction being catalysed by a separate enzyme: A à B à C à D à E a b c d If each enzyme obeys only Michaelis-Menten kinetics, i.e changes its rate of activity in proportion to the concentration of substrate, up to Vmax at saturating levels of [S], then all A will be converted to E through the pathway at a steady rate, until there is no more A. As B is formed it is immediately acted upon by enzyme b and converted to C, and so on.

23. Let us now suppose thatEis not being used by the cell at the moment, and it accumulates to the degree that it starts to force reaction d into reverse, causing a build up ofD. A à B à C à D à E a b c d

24. Let us now suppose thatEis not required by the cell at the moment, and it accumulates to the degree that it starts to force reaction d into reverse, causing a build up ofD. A à B à C à D à E a b c d This effect will slowly work its way back through the system until B starts to accumulate. The end result will be that varying levels of all of the intermediate molecules in the pathway will be present in the cell. This will not matter unlessB,CorDare inherently dangerous as inhibitors of other reactions, which they often are, Or if the molecules ofAwasted in the formation of unwantedB, C, D&Ecould have been put to better use elsewhere.

25. …if the molecules of A wasted in the formation of unwanted B, C, D & E could have been put to better use elsewhere, such as Making U and J. Q à R à S à T à U A à B à C à D à E a b c d F à G à H à I à J

26. The most efficient way to prevent such wastage is a feed-back control loop: when the correct level of E has been attained, the whole pathway is switched off. Q à R à S à T à U A à B à C à D à E a b c d F à G à H à I à J This is the principle operating in allosteric mechanisms for the control of metabolism intracellularly, or within an intracellular compartment, such as an organelle.

27. Allosteric enzymes have a site apart from the substrate binding/active site to which activity modulating molecules bind. The binding of these modulators causes a change in the 3D structure of the substrate binding/active site. This change can increase or decrease the rate of catalysis, depending on whether the modulator is positive (a stimulator) or negative (an inhibitor).

28. In the case of the glycolytic pathway, the purpose of which is to provide the cell with ATP, the allosteric modulator is ATP. The key regulatory enzyme of glycolysis is phosphofructokinase 1 (PFK 1) which has two ATP binding sites: one for the substrate ATP and the allosteric (from the Greek meaning “other site”) site to which ATP binds when the intracellular concentration has reached a high enough level. Thus ATP controls its own formation,and when it is sufficient, no more glucose is broken down by this pathway allowing its use in other pathways, or for storage.

30. The control of glycolysis occurs mainly at PFK1, with some inhibition of pyruvate kinase by ATP in some tissues.

31. The full picture of the control of glycolysis naturally involves input from metabolically related pathways, such as glycogen synthesis and breakdown, fatty acid synthesis and breakdown, etc. From Matthews & van Holde

32. From Voet & Voet

33. Regulatory enzymes can be thought of as having two stable conformations: tense and relaxed. From Campbell Within their metabolic compartment the two forms are in dynamic equilibrium, with an equilibrium constant that varies according to the enzyme.

34. The two conformations have different substrate binding affinities, as well as different catalytic activites. From Campbell

35. Most regulatory enzymes have more than one identical catalytic subunit, and often have a complex quaternary structure. From Campbell

37. The mechanism by which allosteric enzymes exert control over metabolic pathways allows for very fine control within metabolic compartments: as levels of negative modulators drop, more of the tense conformation enzymes are allowed to assume the relaxed, more active conformation and catalysis speeds up until the concentration of modulator rises again to shut off its own production.

38. A slightly different strategy is employed when a force external to the cell exerts control over intracellular metabolic pathways, such as occurs when a hormone binds to its receptor on the external surface of the plasma membrane: Covalent modification – often the attachment of a phosphate group - of a number of key regulatory enzymes causing them to change from tense to relaxed conformations, or vice versa.

40. When blood glucose is low, liver & muscle cells do not want to store glucose as glycogen, so glycogen synthase is inhibited.

41. This enzyme mobilises the glucose stored in glycogen by phosphorylating the glucose residues at the ends of all branches. When blood glucose is low the liver is required to break down its glycogen and export the glucose rapidly for the brain to use.