Enzymes and enzymes inhibition, GPCR

This power-point presentation will give a complete overview about enzymes, nomenclature of enzymes. Enzymes inhibition is also covered in this ppt. Along with some basin introduction to G- protein coupled receptors is also provided.

1. Enzymes and Enzymes Inhibition, G-Protein-Coupled receptors Presented by: Abhishek .S. Joshi First Year M. Pharm (Sem 2) (Pharmacology) SSR College of Pharmacy, Silvassa

2. Enzymes

3. Introduction to Enzymes Enzymes are biocatalysts - the catalysts of life. A catalyst is defined as a substance that increases the velocity or rate of a chemical reaction without itself undergoing any change in the overall process. Enzymes may be defined as biocatalysts synthesized by living cells. They are protein in nature (exception - RNA acting as ribozyme), colloidal and thermolabile in character, and specific in their action. For example, In the laboratory, hydrolysis of proteins by a strong acid at 100'C takes at least a couple of days. The same protein is fully digested by the enzymes in gastrointestinal tract at body temperature (37'C) within a couple of hours. This remarkable difference in the chemical reactions taking place in the living system is exclusively due to enzymes. History: Berzelius in 1836 coined the term catalysis “ Greek: to dissolve”. In 1878, Kuhne used the word enzyme “ Greek: in yeast ”. Isolation of enzyme system from cell-free extract of yeast was achieved in 1883 by Buchner. Sumner first achieved the isolation and crystallization of the enzyme urease from jack bean and identified it as a protein.

4. Nomenclature and Classification of Enzymes

5. Previously, enzymes were given by their discoverers in an arbitrary manner. Sometimes, the suffix- ase was added to the substrate for naming the enzymes e.g. lipase acts on lipids. These are known as trivial names of the enzymes which, however, fail to give complete information of enzyme reaction. The International Union of Biochemistry ( lUB ) appointed an Enzyme Commission in 1961 . The committee made a thorough study of the existing enzymes and devised some basic principles for the classification and nomenclature of enzymes. IUB classified the enzymes into 6 major classes.

6. Oxidoreductases : Enzymes involved in oxidation-reduction reactions. Transferases :Enzymes that catalyze the transfer of functional groups. Hydrolases : Enzymes that bring about hydrolysis of various compounds. Lyases : Enzymes specialized in the addition or removal of water, ammonia, CO2 etc. Isomerases :Enzymes involved in all isomerization reactions. Ligases : Enzymes catalyzing the synthetic: Reactions (Greek : ligate-to bind) where two molecules are joined together and ATP is used.

7. Chemical nature and properties of enzymes The functional unit of the enzyme is known as holoenzyme which is often made up of apoenzyme (the protein part) and a coenzyme (non-protein organic part) Holoenzyme Apoenzyme + Coenzyme (active enzyme) (protein part) (non-protein part) The term prosthetic group is used when the non-protein moiety tightly (covalently) binds with the apoenzyme. The word monomeric enzyme is used if it is made up of a single polypeptide e.g. ribonuclease, trypsin. Some of the enzymes which possess more than one polypeptide (subunit) chain are known as oligomeric enzymes e.g. lactate dehydrogenase, aspartate transcarboxylase etc.

8. Factors affecting enzyme activity Concentration of enzyme- As the concentration of the enzyme is increased, the velocity of the reaction proportionately increases. Concentration of substrate- Increase in the substrate concentration gradually increases the velocity of enzyme reaction within the limited range of substrate levels. Effect of temperature- Velocity of an enzyme reaction increases with increase in temperature up to a maximum and then declines. A bell-shaped curve is usually observed. Effect of p H- Increase in the hydrogen ion concentration (pH) considerably influences the enzyme activity and a bell-shaped curve is normally obtained. Effect of product concentration- The accumulation of reaction products generally decreases the enzyme velocity. Effect of activators - Some of the enzymes require certain inorganic metallic cations like Mg2+, Mn2+, zn2+, ca2+, co2*, cu2+, Na+, K+ for their optimum activity. Metals function as activators of enzyme velocity and bring about conformational changes. Effect of time - Under ideal and optimal conditions (like pH, temperature etc.), the time required for an enzyme reaction is less. Effect of light and radiation- Exposure of enzymes to ultraviolet, beta, gamma and X-rays inactivates certain enzymes due to the formation of peroxides.

9. Mechanism of Enzyme action

10. Theories of mechanism of enzyme action Lock and Key model Or Fischer's template theory Induced fit theory or Koshland's model Substrate strain theory

11. Enzyme Inhibition

12. Enzyme inhibitor is defined as a substance which binds with the enzyme and brings about a decrease in catalytic activity of that enzyme. The inhibitor may be organic or inorganic in nature. There are three broad categories of enzyme inhibition. Reversible inhibition. Irreversible inhibition. Allosteric inhibition

13. Reversible inhibition The inhibitor binds non-covalently with enzyme. The enzyme inhibition can be reversed if the inhibitor is removed. The reversible inhibition is further sub-divided into l. Competitive inhibition ll. Non-competitive inhibition Competitive Inhibition: The inhibitor which closely resembles the real substrate is regarded as a “substrate analogue” The inhibitor competes with substrate and binds at the active site of the enzyme but does not undergo any catalysis Eg. Methanol is toxic to the body when it is converted to formaldehyde by the enzyme alcohol dehydrogenase (ADH). Ethanol can compete with methanol for ADH. Thus, ethanol can be used in the treatment of methanol poisoning.

14. Another example of competitive reversible inhibition: Antimetabolites (5-fluorouracil) are the chemical compounds that block the metabolic reactions by their inhibitory action on enzymes. Antimetabolites are usually structural analogues of substrates and thus are competitive inhibitor. Non competitive inhibition The inhibitor binds at a site other than the active site on the enzyme surface . This binding impairs the enzyme function. The inhibitor has no structural resemblance with the substrate. (A) Competitive Inhibition (B) Non-competitive inhibition.

15. Irreversible inhibition The inhibitors bind covalently with the enzymes and inactivate them, which is irreversible. These inhibitors are usually toxic poisonous substances. Examples: Iodoacetate is an irreversible inhibitor of the enzymes such as papain and glyceraldehyde 3-phosphate dehydrogenase . Iodoacetate combines with sulfhydryl (-SH) groups at the active site of these enzymes and makes them inactive. Di-isopropyl fluorophosphate (DFP) is a nerve gas developed by the Germans during Second World War. DFP irreversibly binds with enzymes containing serine at the active site, e.g. serine proteases, acetylcholine esterase. Many organophosphorus insecticides like melathion are toxic to animals (including man) as they block the activity of acetylcholine esterase, resulting in paralysis of vital body functions.

16. Alcohol gets metabolized by aldehyde dehydrogenase. Aldehyde that is produced gets converted to acetic acid. Disulfiram irreversibly inhibits enzyme aldehyde dehydrogenase. Thus accumulation of aldehyde makes a person sick and thus alcohol consumption is stopped. The penicillin antibiotics act as irreversible inhibitors of serine - containing enzymes, and block the bacterial cell wall synthesis.

17. Suicide inhibition: Specialized form of irreversible inhibition. Original inhibitor (structural analogue or competitive inhibitor) is converted to more potent form by the same enzyme that is to be inhibited. The so formed inhibitor binds irreversibly with the enzyme. A good example of suicide inhibition is allopurinol (used in the treatment of gout) Allopurinol, an inhibitor of xanthine oxidase, gets converted to alloxanthin, a more effective inhibitor of this enzyme.

18. Allosteric Inhibition Allosteric inhibition is the slowing down of enzyme-catalyzed chemical reactions that occur in cells. These metabolic processes are responsible for the proper functioning and maintenance of our bodies’ equilibrium, and allosteric inhibition can help regulate these processes. Allosteric inhibitors slow down enzymatic activity by deactivating the enzyme. An allosteric inhibitor is a  molecule  that binds to the enzyme at an  allosteric site.  This site is not at the same location as the active site. Upon binding with the inhibitor, the enzyme changes its 3D shape.  It is a form of  noncompetitive inhibition Allosteric inhibitors prevent the body from wasting energy to create unnecessary products.  Examples: In glycolysis (cellular respiration), enzyme Phosphofructokinase is responsible for conversion of ADP to ATP. Once there is an excess of ATP in the system, ATP acts as allosteric inhibitor. It binds to phosphofructokinase to slow down the conversion of ATP. In this way, ATP is preventing the unnecessary production of itself.

19. G-Protein Coupled Receptors

20. They are membrane receptors that are coupled to intracellular effector systems via a G-protein . After stimulation of these receptor, action is produced within seconds They constitute the largest family, and include receptors for many hormones and slow transmitters. For example the muscarinic acetylcholine receptor, adrenergic receptors and chemokine receptors.

21. Molecular Structure G-protein-coupled receptors consist of a single polypeptide chain of up to 1100 residues. Their characteristic structure comprises seven transmembrane α helices , similar to those of the ion channels, with an extracellular N-terminal domain of varying length, and an intracellular C-terminal domain. GPCRs are divided into three distinct families. They share the same seven-helix ( heptahelical ) structure, but differ in other respects, principally in the length of the extracellular N terminus and the location of the agonist binding domain .

22. G-Protein G-proteins comprise a family of membrane-resident proteins whose function is to recognize activated GPCRs and pass on the message to the effector systems that generate a cellular response. They actually called G-proteins because of their interaction with the guanine nucleotides, GTP and GDP. Structure : G-proteins consist of three subunits: α, β and γ. Guanine nucleotides bind to the α subunit, which has enzymic activity, catalyzing the conversion of GTP to GDP. The β and γ subunits remain together as a βγ complex. All three subunits are anchored to the membrane through a fatty acid chain, coupled to the G-protein through a reaction known as prenylation

23. Step involved in action of G protein In the 'resting' state, the G-protein exists as an unattached αβγ trimer , with GDP occupying the site on the α subunit. When a GPCR is activated by an agonist molecule, a conformational change occurs , involving the cytoplasmic domain of the receptor, causing it to acquire high affinity for αβγ . Association of αβγ with the receptor causes the bound GDP to dissociate and to be replaced with GTP (GDP-GTP exchange), which in turn causes dissociation of the G-protein trimer , releasing α-GTP and βγ subunits; these are the 'active' forms of which diffuse in the membrane and can associate with various enzymes and ion channels, causing activation of the target . Association of α subunits with target enzymes can cause either activation or inhibition , depending on which G protein in involved. the G-protein, Signaling is terminated when the hydrolysis of GTP to GDP occurs through the GTPase activity of the α subunit. The resulting α-GDP then dissociates from the effector, and reunites with βγ, completing the cycle. Four main classes of G-protein (G s , G i, G o and G q ) are of pharmacological importance.

24. Targets for G-Proteins The main targets for G-proteins, through which GPCRs control different aspects of cell function Adenylyl cyclase , the enzyme responsible for cAMP formation Phospholipase C , the enzyme responsible for inositol phosphate and diacylglycerol (DAG) formation Ion channels , particularly calcium and potassium channels Rho A/Rho kinase , a system that controls the activity of many signaling pathways controlling cell growth and proliferation, smooth muscle contraction, etc.