MORE ON ENZYMES

1. MORE ON ENZYMES JULIUS P. MARIO, RMT, MS

2. ENZYME REGULATION • Feedback Control • Proenzymes • Allosterism • Protein Modification • Isoenzymes

3. FEEDBACK CONTROL • Formation of the product inhibits an earlier reaction in a sequence • In the reaction, E1 E2 E3 A  B  C  D • D may inhibit E1 activity by competitive or noncompetitive inhibition • When D is low, all three reactions proceed rapidly • When D is high, E1 becomes inhibited

4. PROENZYMES • Inactive form of an enzyme • Also known as zymogen • Activated by cleavage of excess polypeptide chain • Cleavage of peptide promotes structural changes, thus, functional • Examples are • Trypsinogen = cleavage of the 6 amino acids from the N-terminal by enteropeptidase converts it to active trypsin • Angiotensinogen = cleavage of the last 2 amino acids from the C-terminal by ACE converts it to a vasoactive octapeptide, angiotensin

5. PROENZYMES • Chymotrypsinogen = cleavage of the 245 residues long polypeptide between R-15 and I-16 from the N-terminal by trypsin converts it to active п-chymotrypsin but the fully active form is α-chymotrypsin

6. PROENZYMES • Some enzymes have proenzymes because they are highly destructive when produced directly in their active forms • Should these leak or exist in excess, inhibitory proteins bind them to render them inactive

7. ALLOSTERISM • If a substance binds noncovalently and reversibly to a site other than the active site, • It may inhibit the enzyme (Negative modulation) • It may stimulate enzyme action (Positive modulation)

8. ALLOSTERISM • Allo means other; steric means shape • Possible conformational changes affect the behavior of proteins • Due to multiple forms of the quaternary structure of some allosteric enzymes • A substance that modifies 4o structure and thus, affect behavior is an allosteric effector • Allosteric effectors are substrates, activators, and inhibitors

9. ALLOSTERIC PROTEINS • Proteins in which subtle changes at one site affects the structure and function of another site • Due to cooperative effects • Depicted as sigmoidal curve on enzyme kinetics • Examples are Aspartate transcarbamoylase (ATCase) & Hemoglobin

10. NONALLOSTERIC PROTEINS • At first, dependent on the [S] with a maximal rate not dependent on [S] anymore • Depicted as a hyperbolic curve on Michaelis-Menten kinetics • Examples are chymotrypsin & myoglobin

11. HOMOTROPIC vs. HETEROTROPICEffects • Homotropic effects are allosteric interactions that occur when several identical molecules are bound to a protein. • Heterotropic effects are allosteric interactions that occur when different substances are bound to a protein.

12. PROTEIN MODIFICATION • Modification is usually a change in the primary structure, typically by the addition of a functional group covalently bound to the apoenzyme • Best-known example is the activation/inhibition of phosphorylation • Glycogen phosphorylaseis active when it is phosphorylated at its serine or tyrosine residue • Pyruvate kinasefrom the liver is inactive when it is phosphorylated

13. ISOENZYMES • Enzymes that perform the same function but have different combinations of subunits, thus have different 4o structures • Have different electrophoretic mobilities, Km, and origins • Act on the same substrate • LDH has 4 subunits • LD1 (H4), LD2 (H3M), LD3 (H2M2), LD4 (HM3) & LD5 (M4)

14. CARBOXYPEPTIDASES • Cleave peptides and proteins at the carboxyl terminal of a particular amino acid in the chain • Trypsin = at lysyl or arginyl • Chymotrypsin = at phenylalanyl, tryptophanyl or tyrosyl as well as L, H & Q • Pepsin = same with chymotrypsin and others • Thermolysin = at isoleucyl, leucyl or valyl

15. PROTEASE FAMILY • All members have similar chemical form • Serine Proteases = cleaves at seryl residues (chymotrypsin, trypsin and elastin) • Cysteine protease = cleaves at cysteinyl residue (papain, a meat tenderizer) • Aspartyl protease = a pair of aspartate side chains, sometimes on different subunits participate in the reaction (pepsin and HIV protease)

16. ABZYMES • Antibodies elicited by antigenic proteins • Designer enzymes which can catalyze a wide variety of reactions • Usually are transition-state analogs which when introduced into the body becomes immunogenic • Nα-(5’-phosphopyridoxyl)-L-lysine is the transition-state analog for the reaction between an amino acid and pyridoxal-5’-phosphate

17. CELLULAR LEVEL • SER enzymes • Detoxification by the cytochrome P-450 enzymes • Lipid synthesis & degradation by • Cytosolic side = cyt b5, cyt b5 reductase, NADPH-ferrihemoprotein reductase, ATPase, 5’-NT, glycolipid mannosyl transferases, hydroxymethylglutaryl-CoA reductase, some 30 enzymes for steroid synthesis and 20 enzymes of lipid synthesis • Luminal side = cyt P-450, cyt P-450 reductase, glucose-6-phosphatase and β-glucuronidase

18. RER • Transport enzymes for glycosylation of newly formed proteins; flippases for phospholipid synthesis • GOLGI BODY • Enzymes for posttranslational modifications of proteins synthesized on membranes as well as recycling of membrane material; 5’-NT, NADH:cyt c oxidoreductase, NADPH:cyt c reductase, UDP-galactose-N-acetylglucosamine-β-D-galactosyltransferase, and many glycosylation enzymes

19. LYSOSOMES • Hydrolytic enzymes such as proteinases, glycosidases, lipases, phosphatases, nucleases, and sulfatases • MICROBODIES • Mainly oxidative enzymes • VACUOLES • V-type H+-translocating ATPase

20. BRUSH BORDER ENZYMES • Disaccharidases found on the microvilli of the small intestine • Once lost, can be re-synthesized via enzyme induction • Substrates commonly acted upon are maltose, sucrose, and lactose

21. ENZYMES AS VIRULENCE FACTORS • Helicobacter pylorisurvives the harsh gastric milieu by the urease on its cell wall. The ammonia produced counters the harmful effects of the acid. • Some pathogenic cocci are capable of producing hemolysins which interfere with oxygen transport in an organism

22. EFFECTS ON ENZYMES • Bactericidal antibiotics for both Gram positive and Gram negative organisms act via inhibition of cell wall synthesizing protein synthase (e.g. Carbapenems) • Binds to the β subunit of RNA polymerase to inhibit transcription of mostly Gram+ bacteria & Mycobacteria (e.g. Rifampin)

23. An example of a medicinal enzyme inhibitor is sildenafil (Viagra), a common treatment for male erectile dysfunction. • This compound is a potent inhibitor of cGMP specific phosphodiesterase type 5, the enzyme that degrades the signalling molecule cyclic guanosine monophosphate. • This signalling molecule triggers smooth muscle relaxation and allows blood flow into the corpus cavernosum, which causes an erection. Since the drug decreases the activity of the enzyme that halts the signal, it makes this signal last for a longer period of time.

24. Another example of the structural similarity of some inhibitors to the substrates of the enzymes they target is seen in the figure comparing the drug methotrexate to folic acid. Folic acid is the oxidised form of the substrate of dihydrofolate reductase, an enzyme that is potently inhibited by methotrexate. Methotrexate blocks the action of dihydrofolate reductase and thereby halts thymidine biosynthesis. This block of nucleotide biosynthesis is selectively toxic to rapidly growing cells, therefore methotrexate is often used in cancer chemotherapy.

25. Drugs also are used to inhibit enzymes needed for the survival of pathogens. For example, bacteria are surrounded by a thick cell wall made of a net-like polymer called peptidoglycan. Many antibiotics such as penicillin and vancomycin inhibit the enzymes (the transpeptidase from the bacteria Streptomyces R61) that produce and then cross-link the strands of this polymer together. This causes the cell wall to lose strength and the bacteria to burst.

26. Drug design is facilitated when an enzyme that is essential to the pathogen's survival is absent or very different in humans. • Humans do not make peptidoglycan, therefore inhibitors of this process are selectively toxic to bacteria. • Selective toxicity is also produced in antibiotics by exploiting differences in the structure of the ribosomes in bacteria, or how they make fatty acids

27. AcCHE, an enzyme found in animals from insects to humans. It is essential to nerve cell function through its mechanism of breaking down the neurotransmitter acetylcholine into its constituents, acetate and choline. • This is somewhat unique among neurotransmitters as most, including serotonin, dopamine, and norepinephrine, are absorbed from the synaptic cleft rather than cleaved. • Reversible competitive inhibitors, such as edrophonium, physostigmine, and neostigmine, are used in the treatment of myasthenia gravis and in anaesthesia. The carbamate pesticides are also examples of reversible AChE inhibitors. • The organophosphate insecticides such as malathion, parathion, and chlorpyrifosirreversibly inhibit acetylcholinesterase.

28. ENZYME DEFICIENCIES

29. MUCOPOLYSACCHARIDOSIS MPS Type Eponym Deficient Enzyme I H Hurler a-iduronidase I H/S Hurler-Scheie a-iduronidase I S Scheie a-iduronidase II Hunter Iduronidase sulfatase III‡ Sanfilippo A Heparan sulfatase Sanfilippo B N-acetylglucosaminidase Sanfilippo C Acetyl CoA glucosamine acetyltransferase Sanfilippo D N-acetylglucosamine-6-sulfatase IV Morquio A Galactosamine-6-sulfatase Morquio B b-galactosidase V Nonexistent VI Maroteaux-Lamy N-acetylhexosamine-4-sulfatase VII Sly b-glucuronidase IX Hyaluronidase Hyaluronidase Deficiency

30. GLYCOGEN STORAGE DISEASES Type - Enzyme Deficient -Clinical Features I (von Gierke) - Liver and kidney Glucose-6-phosphatase Hepatomegaly, lactic acidosis, hyperlipidemia, severe fasting hypoglycemia II (Pompe)- All tissues’ alpha 1,4-glucosidase Cardiomegaly, muscle weakness, death in infancy and adults III (Cori-Forbes)- All tissues’ debrancher enzyme Hepatomgaly, muscle weakness, fasting hypoglycermia IV (Andersen)- All tissues’ brancher enzyme Portal cirrhosis, death in infancy V (McArdle)- Muscle phosphorylase Pain and stiffness after exertion; myoglobinuria

32. LIPIDOSES a group of inherited disorders characterized by the accumulation of lipids in tissuesespecially the brain due to deficiency in a particular sphingolipid catabolic enzyme

33. Niemann-Pick disease • Deficiency in sphingomyelinase and accumulation of sphingomyelin Gaucher’s disease • Deficiency in -D-glucosidase and accumulation of glucocerebroside

34. Krabbe’s disease • Deficiency in -D-galactosidase and accumulation ofgalactocerebsides Fabry’s disease • Deficiency in -D-galactosidase and accumulation of ceramide trihexoside

35. Tay-Sach’s disease • Deficiency in -D-hexaminidase A and accumulation of ganglioside GM2 Metachromatic Leukodystrophy • Deficiency in sulfatide sulfatase and accumulation of-sulfogalactocerebroside