Biosynthesis of Enzymes They must be synthesized when needed

1. Biosynthesis of Enzymes • They must be • synthesized when needed • transported to appropriate cellular location • degraded when no longer needed • Research in ribosome structure reveals that proteins are synthesized by a gigantic RNA enzyme (ribozyme)

3. Wobble • When several codons encode same amino acid, the difference is usually in the third position • If an anticodon recognize a codon as a triplet, there should be tRNAs for each codon • But some anticodons of tRNAs contain inosinate (I) • It forms rather weak bonds than Watson-Crick base pairs

4. Wobble hypothesis • First 2 bases of codon form strong bonds and determine the specificity • First base of anticodon determines the number of codons recognized by tRNA • The wobble (or third) base of the codon also permits rapid dissociation of tRNA during protein synthesis • So accuracy and speed of reaction is balanced by this way

5. Protein Synthesis • Activation of amino acids: amino acids are attached to corresponding tRNA by aminoacyl-tRNA synthetase (ATP) • Initiation: mRNA binds to small subunit of ribosome and initiating tRNA, then large subunit binds -> initiation complex (GTP, initiation factors) • Elongation: succesive covalent attachment of amino acids (GTP, elongation factors) • Termination and release: completion is signaled by a termination codon (release factors) • Folding and posttranslational processing: removal of one or more amino acid, addition of acetyl, phosphoryl, methyl, carboxyl or other groups, attachment of oligosccharides or prosthetic groups

6. Activation and Initiation • Bacterial ribosome have 3 sites to bind aminoacyl-tRNAs: • aminoacyl (A) site • peptidyl (P) site • exit (E) site

7. Elongation

8. Termination

9. Polypeptide chains undergo folding and processing • Post-translational modifications: • Amino or carboxy terminal modifications (amino terminal Met residue is generally removed) • Loss of signal sequences (sequences to direct polypeptide to its ultimate location) • Modification of individual amino acids: phosphorylation, carboxyl group addition, methylation • Attachment of carbohydrates • Addition of isophenyl groups (helps to anchor protein to the membrane) • Addition of prosthetic groups (e.g. heme group) • Proteolytic processing (from large inactive precursors...) • Formation of disulphide xlinks (protect from denaturation)

10. Protein synthesis is inhibited by many antibiotics and toxins Puromycin: binds to A site, participate in peptide bond formation and dissociate with unfininished peptide... • Some other antibiotics: • Tetracyclins (block A site), • Chloramphenicol (block peptidyl transferase), • Streptomycin (dose dependent action) Toxins (to mammalians): Diphtheria toxin and ricin (from castor bean)

11. Specificity of enzyme action-1 • Most enzymes are highly specific to their substrate and reaction catalysed • Bond specificity: e.g peptidase, phosphatase • Group specificity: e.g hexokinase • Absolute or near-absolute specificity:e.g. glucokinase • Stereospecificity: • Dehydrogenases catalyst the transfer of hydrogen from the substrate to a particular side of nicotinamide ring in NAD+ or NADP+ • Phenylalanine hydroxylase uses L-Phe not D-Phe • NZ-catalysed rxns may yield stereospecific products even when substrate possesses no asymmetric carbon atom... • Importance of specificity in DNA replication and protein synthesis • proofreading

12. Specificity of enzyme action-2 The active site • Orgston (1948)  at least three different points of interaction... • These interactions can have: • Binding function  binding sites • Catalytic function  catalytic sites • A.acid residues in the active site which do not have either of these functions may also contribute to specificity... • Side-chains must be of suitable size, shape and character • Creation of a specific microenvironment...

13. Specificity of enzyme action-3 The active site • The active site takes up a relatively small portion of the total volume of an enzyme • The active site is a 3-D entity • In lysozyme, the important groups in the active site are contributed by residues numbered 35, 52, 62, 63, and 101 in their linear sequence of 129 a.acids • The specificity of binding depends on the precisely defined arrangement of atoms in an active site • Substrates are bound to enzymes by relatively weak forces • Active sites are clefts or crevices • In all enzymes of known structure, substrate molecules are bound to a cleft or crevice from which water is largely excluded

14. Specificity of enzyme action-4 The Fischer “lock-and-key” hypothesis • Fischer (1890) • According to this model, structures do not change their shape during the binding process The Koshland “induced-fit” hypothesis • Koshland (1958) • X-ray diffraction analysis and NMR data have revealed differences in structure btw free and substrate-bound NZs • Conformational change • Such mechanism could help to achieve high degree of specificity.... • e.g. yeast hexokinase D-hexose + ATP D-hexose-6-P + ADP

15. Induced-fit

16. Specificity of enzyme action-5 Hypothesis involving strain or transition-state stabilization • Both lock-and-key and induced-fit can explain NZ specificity BUT what is the mechanism to drive the reaction??? • Energy is often spent in substrate-binding and further energy must be supplied for reaction to proceed • Haldane (1930) and Pauling (1948)  Binding energy is used to distort the substrate in such a way to facillate the subsequent reaction • Little clear-cut evidence on distorted binding • Transition state stabilization is more likely • Substrate is bound in an undistorted form, but ES complex possesses various unfavorable interaction yielding: ES complex  transition state  products

17. Specificity of enzyme action-6 Specificity of enzymes and drug design • In addition to exploiting a binding event, a highly specific chemical events takes place in the enzyme's active site • There are 317 FDA approved drugs that target enzymes (human, bacterial, viral, fungal and protazoal) • The majority of these drugs (~65%) rely on some type of substrate mimicking (direct interaction with cofactor, structural resemblance to substrate, transition state analogue, etc.). • In order to exploit these highly specific chemical interactions, one must fully understand the enzyme mechanism, and perhaps even obtain a detailed transition state structure

18. Monomeric and oligomeric enzymes-1 Monomeric Enzymes • Only a single polypeptide chain.... • Very few monomeric NZs are known and all catalyse hydrolytic rxns (e.g. Proteases) • 100-300 a-acid with Mw: 13-35 kDa • Most act without a cofactor • They are often synthesized in inactive form: proenzyme or zymogen

19. Monomeric and oligomeric enzymes-2 The serine proteases: • Chymotrypsin, trypsin and elastase • Only 40% of the 1O structure is common but • Most catalytically important residues are same • Their 3O structures are very similar • All are endopeptidases but their specificity different: • Chymotrypsin: hydrophobic side chains • Trypsin: specificity for basic side chains • Elastase: small non-polar side chains Some other monomeric NZs • Pepsin (an acid protease) in mammals • Thiol proteases (e.g. papain, ficin) in plants • Exopeptidases (e.g. carboxypeptidase A and B) • Ribonucleases and lysozymes (act on non-protein substrates...)

20. Monomeric and oligomeric enzymes-3 Oligomeric Enzymes • Two or more polypeptide chains • Linked usually by non-covalent interactions and NEVER by peptide bond • May be identical or different (subunits) • Mw >35 kDa • Not synthesized as inactive zymogens

21. Monomeric and oligomeric enzymes-4 Some examples Lactate dehydrogenase (LDH): • Tetramer catalysing the rxn: Lactate + NAD+ pyruvate + NADH + H+ • A tetramer of 140 kDa • Two subunit types (from seperate genes), M- and H-form, predominating different tissues • Monomers are catalytically inactive • All five isoenzymes of LDH can exist...... • They all catalyse the same reaction but with different characteristics

22. Monomeric and oligomeric enzymes-5 Lactose synthase • Non-functional subunit modifies behavior of the functional subunit -lactalbumin + galactosyl transferase • (-) -lactalbumin  involve in the synthesis of the carbohydrate components of glycoproteins (+) -lactalbumin  lactose production

23. Monomeric and oligomeric enzymes-6 Tryptophan synthase • Two different functional subunits (2  and ): indole-3-glycerol-P  indole + glyceraldehyde-3-P ( subunit) indole + L-serine  L-tryptophan ( subunit) • The rate of these partial reactions are less than 5% of the rate of the reaction catalysed by intact 22 enzyme... Pyruvate dehydrogenase • A multienzyme complex.... • Enable pyruvate to enter the TCA cycle  acetyl-CoA production • E.coli enzyme: 60 polypeptide chains with a MW: ca 4600 kDa.... • Three catalytic activities are present

24. Engineered enzymes-1 AIM • to alter the reaction mechanism of the enzyme to catalyse new reactions • to expand substrate specificity • to switch or increase substrate specificity, such as improvement of the enantioselectivity • to increase stability • Alteration of enzyme reaction chemistry is an attractive strategy to obtain new catalysts for the synthesis of fine chemicals

25. Engineered enzymes-2 Directed evolution: • It is a powerful strategy that does not rely on knowledge of the 3-D structure of the enzyme • Usage of molecular biological methods such as random mutagenesis or gene shuffling for generating large diverse DNA libraries • Gene expression • High-throughput screening for the identification of highly selective mutant enzymes Rational redesign: • a good 3-D structure should be available • It is often based on molecular graphics or more advanced molecular modeling (structure-based computational design techniques) • Such models place an emphasis on enthalpic interactions and omit entropic contributions

26. Rational redesign-1 Reshaping enzyme specificity • reshaping a substrate-binding site or cofactor specificity or positioning charged residues to favor one substrate relative to another one Re-engineering catalytic mechanisms • The current view is that chemistry, not binding specificity, is the dominant factor in the evolution of new enzymatic activities. As a consequence, proteins with similar folds can support very different chemical reactions after the incorporation of new catalytic groups Engineering by molecular assembling • assembling the necessary components, that is, the catalytic machinery, a substrate-binding site and so on, on a selected macromolecular template

27. Rational redesign-2 Reshaping enzyme specificity e.g. linoleate 13-lipoxygenase  9-lipoxygenase • by a single substitution, H608V, at the level of the residue that is supposed to be responsible for the positional specificity of the substrate • Bulky His608 replaced  a basic residue at the bottom of the active pocket becomes accessible • The carboxylate group of the lipid substrate, which is normally outside the pocket, becomes able to interact with this basic residue • Different oxygenation site in the lipid

28. Rational redesign-3 Re-engineering catalytic mechanisms • 4 substitutions  an oleate-hydroxylase changed on an oleate-desaturase • Butyrylcholinesterase (BChE) enzyme is transformed to take a potent inhibitor as a substrate • The mechanism of BChE can be redirected to favor the hydrolysis of the strong irreversible inhibitors (e.g. Soman), as opposed to irreversible modification of BChE

29. Rational redesign-4 Engineering by molecular assembling-1 Inventory of robust catalytic machineries • A prerequisite to convergent enzyme redesign is the identification of the small number of catalytic devices that can work in various structural contexts • The TESS software searches through a dataset of PDB structures for user-defined combinations of atoms or residues • The results have been compiled in the PROCAT database of 3-D active site coordinates • Another computational search for consensus catalytic devices, such as the Ser–His–Asp catalytic triad, the His–His heme coordination site

30. Rational redesign-5 Engineering by molecular assembling-2 Searching for appropriate engineering templates • Nature seems to recruit a limited number of protein folds for building a large variety of functions • Some enzyme superfamilies display remarkably divergent properties in terms of functions, while conserving some specific chemical properties • However, the possibilities offered by nature to protein engineers for the redesign of enzyme function seem to go beyond this well-known subset of structures • For example, a soluble and functional chimaeric bacterial–human cytochrome P450 is engineered: The resulting hybrid enzyme exhibits mammalian enzyme active site characteristics, with the solubility property of the bacterial enzyme

31. Rational redesign-6 Engineering by molecular assembling-3 Grafting catalytic machineries • It is possible to engineer catalytic antibodies by grafting functional residues • A protease that is able to cleave a small bacterial protein was engineered from an immunoglobulin single-chain variable fragment after introducing three residues: • a lysine to increase the polarizability of the carbonyl group • a glutamate to increase the nucleophilicity of a nearby water molecule • a histidine to provide a proton to convert the amine into a better leaving group

32. Abzymes-1 • Catalytic antibody, abzyme (mid-80s) • Abzymes which are able to hydrolyze proteins, DNA, RNA, or polysaccharides have been found in the sera of patients with autoimmune and also viral pathologies • Possible applications • As sequence-specific cleaving agents to destroy peptides or carbohydrates associated with viral particles or tumor cells • The genetic deficiency of an extracellular enzyme could be cured by immunization with an appropriate hapten to elicit catalytic antibodies that would substitute the missing enzyme • Catalytic antibodies with the capacity to degrade cocaine into the resulting nontoxic products  for cocain addiction • Detoxification after accidental exposure to insecticides or the intentional poisoning by nerve gas

33. Abzymes-2 • One of the most fascinating potential applications is in the area of prodrug activation in cancer treatment using a strategy called Antibody Directed Abzyme Prodrug Therapy(ADAPT)

34. Ribozymes • In the early 1980s, Sidney Altman and Thomas Cech independently found that RNAs can also act as catalysts for chemical reactions • This class of catalytic RNAs are known as ribozymes (1989 Nobel Prize in Chemistry) • Ribozome • Group I and II introns • «5S rRNA Is a Leadzyme. A Molecular Basis for Lead Toxicity» M.Z. Barciszewska et al (2003) • «Hammerhead RNA motif» In the natural state, is a single strand of RNA: the cleavage is autocatalytic but not a true enzyme in its natural state, as it cannot catalyze multiple turnovers BUT in vitro hammerheads can be engineered • Artificial ribozymes