Dr. Ketki K, MBBS, MD Assistant Professor Department of Biochemistry

1. Dr. Ketki K, MBBS, MDAssistant Professor Department of Biochemistry

3. Regulation of Enzyme Activity

4. Short term regulation • Altering the activity of existing enzyme (fine control) Allosteric regulation: allosteric, feed-back, feed-forward Covalent modification Compartmentalization

5. Long term regulation • Altering concentration of enzyme (coarse regulation) Induction (derepreession) Repression

6. Allosteric regulation • Allosteric enzyme: (oligomers) • It has one catalytic site where substrate binds • Another separate allosteric site where the modifier binds • Modifier can enhance/inhibit the acitivity of enzyme (allosteric activator/positive modifier,allosteric inhibitior/negative modifier) • Binding of substrate to one subunit of enzyme enhances binding by other subunits(Positive cooperativity) • Binding of substrate to one subunit of enzyme decreases binding by other subunits(Negative cooperativity)

8. Salient features of allosteric regulation • Inhibitor is not a substrate analogue • Partially reversible when excess substrate is added • Km is usually increased • Vmax is reduced • Effect of allosteric modifier is maximum at or near substrate concentration equivalent to Km • Most allosteric enzymes have quaternary structure.They are made up of subunits • Body uses allosteric enzymes for regulating metabolic pathways/rate limiting enzymes/key enzymes

9. Classes of allosteric enzymes • Allosteric enzymes • Enzymes which are regulated by allosteric mechanism are c/a allosteric enzymes • Allosteric effectors divided based on Km and Vmax • K-class of allosteric enzymes effector changes Km, not Vmax DBR plot similar to competitive inhibition is obtained Eg: Phosphofructokinase

10. 2) V-class of allosteric enzymes Effector alters Vmax,not the Km DBR plot resembles that of non competitive inhibition Eg: acetyl CoA carboxylase

11. Conformational changes in allosteric enzymes • Oligomeric enzymes • Binding of effector to one subunit brings conformational change in active site of enzyme leading to inhibition or activation of catalytic activity • Allosteric enzyme exists in two states Tense(T) & Relaxed (R)state. • T & R states are in equilibrium T Allosteric activator/substrate R Allosteric inhibitor

12. Allosteric inhibitor favours T state • Allosteric activator favours R state • Concentration of enzymes in R state increases as more substrate/allosteric activator is added.therefore binding of substrate to allosteric enzyme is said to be cooperative

13. Cooperative binding

14. Sigmoid shaped curve is obtained

16. Homotropic effect: Substrate influences substrate binding to catalytic site of enzyme through allosteric mechanism • Heterotropic effect: Allosteric modulator influences substrate binding to catalytic site of enzyme through allosteric mechanism It can be positive/negative

17. Example: (in Glycolysis)

18. Succinyl Co-A ALA Synthase δ amino levulinic acid + Glycine First step in heme biosynthesis/Key enzyme End product heme allosterically inhibit the ALA synthase

19. Examples of allosteric enzymes

20. Feedback regulation • The process of inhibiting the first step by the final product in a series of enzyme catalysed reaction of a metabolic pathway. • A e1 B e2 C e3 D e4 E • A = Initial substrate • B,C,D = Intermediates • E= End product • Final end product E inhibits e1 ,regulates the first step • This is c/a negative feed back regulation (or ) end product inhibition

21. Example Carbamoyl phosphate ATC Carbamoyl aspartate + Pi + Aspartate feedback control Cytidine triphosphate (CTP) End product CTP accumulates, inhibits enzyme ATC by feedback mechanism

22. Positive feed forward activation

23. Covalent modification • Addition of group to enzyme protein by a covalent bond or removal of group by cleaving covalent bond • Activity of enzyme may be increased/decreased by covalent modification • Example: Zymogen activation: activation by break down of one or more peptide bonds Chymotrypsinogen, pepsinogen, plasminogen

24. Activation of Zymogen

25. Phosphorylation/dephosphorylation 2ATP 2 ADP cAMP dependant Protein Kinase Phosphorylase b Phosphorylase a (Inactive) (active) Phosphatase (2Pi)

26. Phosphorylation P OH Protein ActiveInactive SerThr Tyr(His) Kinase phosphorylation Conformational Change dephosphorylastion Phosphatase Glycogen phosphorylase b Glycogen phosphorylase a InactiveActive Juang RH (2004) BCbasics

28. Compartmentalization • Generally synthetic and breakdown pathways are operative in different cellular organelles to achieve maximum economy • Example : Fatty acid synthesis in Cytosol Fatty acid oxidation in Mitochondria

30. Induction & repression • Many rate limiting enzymes have short half lives, it helps in efficient regulation of enzyme levels • There are two types of enzymes: 1) Constitutive enzymes: House keeping enzymes Levels of which are not controlled, remains fairly constant 2) Adaptive enzymes: There concentration increase or decrease as per body needs, are well regulated

31. Induction : used to represent increase synthesis of enzymes in response to certain signal (OR) Turing “on” the switch of the gene Repression: indicates decreased synthesis of enzymes in response to certain signal (OR) Turing “off” the switch of the gene

32. Classical example: 1)Induction of lactose utilizing enzymes in bacteria when media contains lactose in absence of glucose. • There is minimal level of enzyme inside the cell.but in presence of inducer(lactose), level of enzymes will go up to thousand times within hours. • By this mechanism,nutrients are utilized most effectively 2) Glucocorticoids induces transaminases,tryptophan pyrrolase 3) Barbiturates induces ALA Synthase

33. Lac operon explains induction/derepression • Lac operon: Unit of gene expression Includes structural genes, control elements, regulator genes, promoter area, operator area

34. Induction/derepression Example : lactose metabolism

36. Repression • Mechanism by which the presence of excess product of a pathway shuts off the synthesis of key enzyme of that pathway at gene level • Example: Heme synthesis is regulated by repression of ALA synthase,key enzyme of the pathway by heme

40. CLINICAL NOTES • Ann O'Rexia, a 23-year-old woman, 5 ft 7 intch tall, is being treated for anorexia nervosa. She has been gaining weight and is now back to 99 lb from a low of 85 lb. Her blood glucose is still below normal (fasting blood glucose of 72 mg/dL, compared with a normal range of 80 to 100 mg/dL). She complains to her physician that she feels tired when she jogs, and she is concerned that the “extra weight” she has gained is slowing her down. • Ann O'Rexia's physician explained that she had inadequate fuel stores for her exercise program. To jog, her muscles require an increased rate of fuel oxidation to generate the ATP for muscle contraction.

41. The fuels used by muscles for exercise include glucose from muscle glycogen, fatty acids from adipose-tissue triacylglycerols, and blood glucose supplied by liver glycogen. • These fuel stores were depleted during her prolonged bout of starvation. In addition, starvation resulted in the loss of muscle mass as muscle protein was degraded to supply amino acids for other processes, including gluconeogenesis (the synthesis of glucose from amino acids and other noncarbohydrate precursors).

42. One of the fuels used by Ann O'Rexia's skeletal muscles for jogging is glucose, which is converted to glucose 6-phosphate (glucose 6-P) by the enzymes hexokinase (HK) and glucokinase (GK). • Glucose 6-P is metabolized in the pathway of glycolysis to generate ATP. This pathway is feedback-regulated, so that as her muscles use ATP, the rate of glycolysis will increase to generate more ATP. • When Ann O'Rexia jogs, the increased use of ATP for muscle contraction results in an increase of AMP, which allosterically activates both the allosteric enzyme phosphofructokinase-1, the rate-limiting enzyme of glycolysis, and glycogen phosphorylase, the rate-limiting enzyme of glycogenolysis.

43. This is an example of feedback regulation by the ATP/AMP ratio.

44. Unfortunately, her low caloric consumption has not allowed feed-forward activation of the rate-limiting enzymes in her fuel storage pathways, and she has very low glycogen stores. Consequently, she has inadequate fuel stores to supply the increased energy demands of exercise. • Therefore, Ann will need to increase her caloric consumption to rebuild her fuel stores. Her physician helped her calculate the additional amount of calories her jogging program will need, and they discussed which foods she would eat to meet these increased caloric requirements. He also helped her visualize the increase of weight as an increase in strength.

45. The hormone epinephrine (released during stress and exercise) and glucagon (released during fasting) activate the synthesis of cAMP in a number of tissues. cAMP activates protein kinase A. • Because protein kinase A is able to phosphorylate key regulatory enzymes in many pathways, these pathways can be regulated coordinately. • In muscle, for example, glycogen degradation is activated while glycogen synthesis is inhibited. At the same time, fatty-acid release from adipose tissue is activated to provide more fuel for muscle.

46. Thank you