Regulation of Enzyme Activity

1 Regulation of Enzyme Activity Manickam Sugumaran Professor of Biology - Boston Boston, MA 02125. The Theme of This Lecture Regulation of Enzyme Activity at Protein Level. 1. Covalent modification. 2. Noncovalent (allosteric) Regulation 3. Protein degradation (will not be considered). Regulation at Different Levels Transcriptional Translational Protein Regulation Regulation degradation 1 2 Inactive protein 4. Allosteric 5 Regulation Active protein DNA RNA Inactive 2 protein Active protein 3. Translational Regulation Covalent modification Why regulate? In the cell, enzymes do not work alone but often work together in groups. These sets of reactions are called metabolic pathways. Given the fact the enormous amount of energy and resources are dedicated for each pathway to carry out different metabolic functions, the cells have to regulate the activities of the enzymes very precisely.

2 Regulation will allow the changing needs of the cell to meet its energy and resource demands. If a product is available in excess, it could then divert the resources to other needy reactions. If a product is in demand, it could activate the pathway to produce more of the biomolecule that is needed. Thus Regulation is the process by which cells can turn on, turn off, or modulate the activities of various metabolic pathways. Adrenaline Turn off Glycogen You see Adenylate Adenylate cyclase (A) Synthesis cyclase (I). the lion. Glycogen You are ATP Cyclic AMP. Synthase (b). afraid!! Protein kinase A (I) Protein kinase A (A). Start Glycogen running Synthase (a). Phosphorylase Phosphorylase kinase (I) kinase (A). Start Phosphorylase b Phosphorylase a Degrade Glycogen Glycogen Glucose-6-P.

3 And get the ATP from glucose-6-P for running !! Amplification of Enzyme Activity Approximately nmoles of adrenaline per gram of muscle will trigger the synthesis of 25 moles of glucose -1- phosphate per minute per gram of muscle. This represents an amplification of 250,000 fold. Four kinds of Regulation Regulation of Enzyme Activity Allosteric Regulation Proteolytic activation (irreversible covalent modification). Reversible covalent modification Stimulation and Inhibition by control proteins Proteolytic activation This kind of activation is irreversible. Once the protein is activated, the process cannot be reversed. Active protein can only be controlled by other kinds of Regulation , such as inhibition or inactivation. Examples of Proteolytic Activation Zymogen activation.

4 Blood clotting. Complement activation. Prophenoloxidase activation. Inactive hormones to active hormones. Activation mechanism of Chymotrypsin 1 Inactive chymotrypsin 245. Trypsin 1 15 16 -chymotrypsin (active) 245. Ser 14 -Arg 15 chymotrypsin Thr 147-Asn-148. 1 13 16 146 149 245. A Chain B Chain C Chain -chymotrypsin (active). Reversible Covalent Modification A single trigger rapidly switches a whole pathway on or off Examples of reversible covalent modifications Phosphorylation - dephosphorylation Adenylation - deadenylation. Reversible covalent modification - Phosphorylation While running, glycogen phosphorylase Activity is enhanced by phosphorylation. At the same time glycogen synthase Activity is shut off by phosphorylation. Regulation - covalent modification ATP ADP.

5 KINASE. O. OH P. O O. O. ACTIVE Enzyme INACTIVE Enzyme . PHOSPHATASE. Pi H2 O. PHOSPHORYLATION AND DEPHOSPHORYLATION. OF SERINE (THREONINE AND TYROSINE). Stimulation and inhibition by control proteins ACTIVATION OF PROTEIN KINASE BY CYCLIC AMP. R R. cAMP cAMP - Regulatory R R Protein Complex C C C + C. INACTIVE. PROTEIN KINASE ACTIVE PROTEIN. KINASE SUBUNIT. ALLOSTERIC PROTEINS SHOW. SIGMOIDAL KINETIC BEHAVIOR. Normal Protein Allosteric Protein Michaelis Menten Kinetics Sigmoidal kinetics v v [S] mM [S] mM. Effect of activator and Inhibitor + Activator Control + Inhibitor v Activator decreases sigmoidity;. Inhibitor increases sigmoidity [S] mM. For ATCase, ATP is an allosteric activator CTP is an allosteric inhibitor Allosteric enzymes Do not exhibit Michaelis Menten type kinetics.

6 Show sigmoidal kinetic behavior. Must undergo drastic conformational changes upon binding of modulatory ligand. Usually possess catalytic and regulatory subunits. Therefore, they are larger and more complex than non-allosteric enzymes . Allosteric Regulation If the substrate itself is regulator, it is called homotropic interaction. If it is a different ligand, it is called heterotropic interaction (can be an activator or an inhibitor). The sigmoidal graph arises due to cooperativity or subunit interaction. Binding of a ligand causes conformational changes in a subunit that may or may not be transmitted to other subunits. Desensitization ATCase treated with mercurials do not exhibit allosteric kinetics. This desensitization is caused by the separation of catalytic and regulatory subunits by the reaction with mercurials.

7 C. C C 2 C C C. R R R. R C R R pCMB +. C 3 R R. C. ATCase Subunits End Product Inhibition If F is available by some chance, the pathway should be shut off. End product inhibition provides this Regulation X. A B C D E F. Concerted model or symmetry model Substrate T state R state The Enzyme exists only in two states The two states are T (taut or tensed) and R (relaxed). Substrates and activators have great affinity for R state Inhibitors have higher affinity for T state Ligands affect the equilibrium between T and R states While going from one state to the other symmetry must be conserved. Concerted model or symmetry model Substrate T state R state Binding of ligand to one subunit always assists the binding of the same ligand to the next subunit - This means that only positive cooperativity is possible.

8 Heterotropic interactions could be either positive or negative. Sequential model Substrate Substrate T T R T R R. Ligand binding alters the conformation of the Enzyme . Binding of ligand to a subunit alters the conformation of only that subunit. This alteration is transmitted to other subunits by subunit interaction. Therefore, multiple states are possible. While going from one state to the other, symmetry need not be conserved. Sequential model Substrate Substrate T T R T R R. Binding of ligand to one subunit may help or hinder its binding to the other subunit - That is both positive and negative homotropic interaction are possible in this model. Heterotropic interactions could be either positive or negative. In the Symmetry model only positive homotropic interaction is allowed.

9 [S binding always helps another S binding]. Heterotropic interactions could be positive or negative. [S and I binding is negative heterotropic interaction and S and A binding is positive heterotropic interaction]. In the Sequential model, both homotropic and heterotropic interactions could be positive or negative. [That is even S binding to one subunit could inhibit the S binding to the next subunit (negative homotropic interaction is possible here)]. Aspartate transcarbamoylase O NH2. O O NH2 H 2N COOH O. P O O HN COOH. O O. + P. COOH O O. COOH. Carbamoyl Aspartic acid Transition State phosphate O. H 2N NH COOH O NH COOH. P. O O O. COOH COOH. N-Carbamoylaspartate N-(phosphonacetyl)-L-aspartic acid For ATCase, CTP is an allosteric inhibitor. ATP is an allosteric activator.

10 O COOH COOH. O O NH2 ATCase NH2. P + O. O O H 2N COOH N COOH. Pi H. Carbamoyl Aspartic acid N-Carbamoyl aspartate phosphate NH 2. C. N. O O O N. O O O O O. P P P O. O O O. Cytidine triphosphate (CTP) OH OH.