Recombinant DNA II; Enzymes I

1. Recombinant DNA II; Enzymes I Andy HowardIntroductory Biochemistry 5 November 2014 Recombinant II; Enzymes I

2. Recombinant DNA and enzymology • We complete our survey of the way molecular biology works experimentally • We then begin an exploration of what enzymes are and how they operate Recombinant II; Enzymes I

3. Recombinant II Protein-protein interactions: screens Genomics Proteomics Polymerase Chain Reaction Mutagenesis Gene Therapy Enzyme classes Enzyme kinetics Michaelis-Menten kinetics: Derivation What we’ll discuss Recombinant II; Enzymes I

4. Protein-protein interactions • One of the key changes in biochemistry over the last two decades is augmentation of the traditional reductionist approach with a more emergent approach, where interactions among components take precedence over the properties of individual components • Protein-protein interaction studies are the key example of this less determinedly reductionist approach Recombinant II; Enzymes I

5. Two-hybrid screens • Use one protein as bait; screen many candidate proteins to see which one produces a productive interaction with that one • Thousands of partnering relationships have been discovered this way • Some of the results are clearly biologically relevant; others less so Recombinant II; Enzymes I

6. 2-hybrid screen(fig. 12.17) • X is bait, fused to DNA binding domain of GAL4 • Y is target, fused to transcriptional activator portion of GAL4 Recombinant II; Enzymes I

7. Reporter constructs:How to study regulation • Put a regulatory sequenceinto a plasmid upstream ofa reporter gene whose productis easy to measure and visualize • Then as we vary conditions, we can see how much of the reporter gets transcribed • Example: Green Fluorescent Protein, which can be readily quantified based on fluorescent yield GFPAequorea victoria27kDa monomer0.9Å resolution Recombinant II; Enzymes I

8. Genomics • Application of these high-throughput techniques to identification of genetic makeup of entire organisms • First virus was completely sequenced in the late 1970’s • First bacterium: Haemophilus influenzae, 1995 • Now > 100 organisms in every readily available phylum Recombinant II; Enzymes I

9. What’s been sequenced? • Cf. Table 12.1 • This list includes only completed eukaryotic projects with size > 20 MB • One might study multiple individuals within a species Recombinant II; Enzymes I

10. How genomics works • A researcher who wishes to draw general conclusions about structure-function relationships may want to learn the sequence (“primary structure”) of many genes and non-genomic DNA in order to draw sweeping conclusions or build a library of genetic constructs, some of which he will understand and others he won’t Recombinant II; Enzymes I

11. Complete sequencing of a genome • Fragment chromosomes • Shotgun sequencing of fragments • Reconstitution based on overlaps • Cross-checking to compensate for errors • Interpretation Recombinant II; Enzymes I

12. Human genome project • Effort began in late 1980’sto do complete sequencingof the human genome • Methods development wasproceeding rapidly duringthe period in question so it“finished” well ahead of schedule in 1999 • Partly federal, partly private • Related efforts in other countries Time3 Jul 2000 Recombinant II; Enzymes I

13. What’s the point? • Better understanding of both coding and non-coding regions of chromosomes • Identification of specific human genes • Medically significant results • Statistical results (x% are Zn fingers…) • Variability within Homo sapiens or some other sequenced organism by comparing complete sequences or ESTs between individuals Recombinant II; Enzymes I

14. Proteomics • Analysis of the resulting list of expressible (not necessarily expressed!) proteins • Often focuses on changes in expression that arise from changes in environmental conditions or stresses • Often useful to analyze mRNAs along with proteins • Mass spectrometry is a key tool in proteomics Recombinant II; Enzymes I

15. How MS works in proteomics • Cartoon from Science Creative Quarterly at U.British Columbia, 2008 Recombinant II; Enzymes I

16. Amplification • Prokaryotic and eukaryotic cells can, through mitosis, serve as factories to make many copies (> 106 in some cases) of a moderately complex segment of DNA—provided that that segment can be incorporated into a chromosome or a plasmid • This is amplification Recombinant II; Enzymes I

17. Polymerase chain reaction (§12.4) Kary Mullis • This is a biochemical tool that enables incorporation of desired genetic material into a cell’s reproductive cycle in order to amplify it • Start with denatured DNA containing a segment of interest • Include two primers, one for each end of the targeted sequence • The sequence of events is now well-defined after three decades of refinement of the approach Recombinant II; Enzymes I

18. PCR: the procedure • Heat to denature cellular dsDNA and separate the strands • Add the primers (ssDNA) and polymerase • Heat again, then cool enough for ligation • Continue cycling to get many cell divisions ~ 106-fold amplification Recombinant II; Enzymes I

19. Cartoon version Image courtesy nobelprize.org Recombinant II; Enzymes I

20. PCR in practice(fig. 12.18) Recombinant II; Enzymes I

21. RT-PCR • Variant on ordinary PCR: starting point is an RNA probe that can serve as a template for DNA via reverse transcriptase • Once cDNA copy is available, normal PCR dynamics apply Cartoon courtesy Cellular & Molecular Biology group at ncvs.org Recombinant II; Enzymes I

22. Mutagenesis • Procedure through which mutations are introduced into genomic or plasmid DNA • May be used: • To generate diversity • To probe the essentiality of specific genes • To examine particular segments of genes • To alter properties of DNA or its mRNA transcript or a translated protein • To provide information and material for gene therapy Recombinant II; Enzymes I

23. Random mutagenesis • DNA (often locally ssDNA) is exposed to mutagens in order to introduce random mispairings or increase the rate of mispairing during replication • Can involve ionizing radiation • Can involve chemical mutagens: • Error-prone PCR • Using “mutator strains” • Insertion mutagenesis • Ethyl methanesulfonate • Nitrous acid and other nitroso compounds Recombinant II; Enzymes I

24. Site-directed mutagenesis • Specific loci in DNA targeted for alteration • Typically involves excision, addition of altered bases, and religation • Can be accomplished even in eukaryotic cell systems • Many biochemical systems can be systematically probed this way: • To find essential amino acids in expressible proteins • To see which amino acids are important structurally • To examine changes at RNA level Recombinant II; Enzymes I

25. How do we use these tools? • Already discussed significance of complete sequencing efforts • Generally: amplification and expression give us access to and control of biochemical systems that otherwise have to be isolated in their original setting • These methods enable controlled experiments on complex systems Recombinant II; Enzymes I

26. Gene therapy • Cloned variant of deficient gene is inserted into human cells • Can be done via viral or other vector carrying an expression cassette • Maloney murine leukemia virus (MMLV, or retroviral approach) works for cassettes up to 9kbp; depends on integrating the cassette into the patient’s DNA • Adenovirus works up to 7.5 kb: never gets incorporated into host, but simply replicates along with host Recombinant II; Enzymes I

27. Retroviral approach Recombinant II; Enzymes I

28. Adenoviral approach Recombinant II; Enzymes I

29. iClicker quiz, question 1 1. In a yeast 2-hybrid experiment, the bait is fused to • (a) The DNA-binding domain of GAL4 • (b) The transcriptional activator domain of GAL4 • (c) Both of the above • (d) Neither of the above. Recombinant II; Enzymes I

30. iClicker quiz, question 2 2. The human genome contains • (a) 115 MBp • (b) 389 MBp • (c) 3038 MBp • (d) 5373 MBp • (e) None of the above Recombinant II; Enzymes I

31. Enzymes • Okay. Having reminded you that not all proteins are enzymes, we can now zero in on proteinaceous enzymes. • Understanding a bit about enzymes makes it possible for us to characterize the kinetics of biochemical reactions and how they’re controlled. • We need to classify them and get an idea of how they affect the rates of reactions. Recombinant II; Enzymes I

32. Enzymes have 3 features • Catalytic power (they lower G‡) • Specificity • They prefer one substrate over others • Side reactions are minimized • Regulation • Can be sped up or slowed down by inhibitors and accelerators • Other control mechanisms exist Recombinant II; Enzymes I

33. IUBMB Major Enzyme Classes Recombinant II; Enzymes I

34. EC System Porcine pancreatic elastasePDB 3EST 1.65 Å 26kDa monomer • 4-component naming system,sort of like an internet address • Pancreatic elastase: • Category 3: hydrolases • Subcategory 3.4: hydrolases acting on peptide bonds (peptidases) • Sub-subcategory 3.4.21: Serine endopeptidases • Sub-sub-subcategory 3.4.21.36: Pancreatic elastase Recombinant II; Enzymes I

35. Category 1:Oxidoreductases • General reaction:Aox + BredAred + Box • One reactant often a cofactor • Cofactors may be organic(NAD or FAD)or metal ions complexed to proteins • Typical reaction:H-X-OH + NAD+ X=O + NADH + H+ Recombinant II; Enzymes I

36. Category 2:Transferases -keto-glutarate • These catalyze transfers ofgroups like phosphate or amines. • Example:L-alanine + a-ketoglutarate pyruvate + L-glutamate • Kinases are transferases:they transfer a phosphate from ATP to something else pyruvate Recombinant II; Enzymes I

37. Category 3:hydrolases Pyrophosphate(dianionic form) • Water is acceptor of transferred group • Ultrasimple: pyrophosphatase:Pyrophosphate + H2O2 Phosphate • Proteases,many other sub-categories Recombinant II; Enzymes I

38. C=C Category 4:Lyases • Non-hydrolytic, nonoxidative elimination (or addition) reactions • Addition across a double bond or reverse • Example: pyruvate decarboxylase:pyruvate + H+ acetaldehyde + CO2 • More typical lyases add across C=C Recombinant II; Enzymes I

39. Category 5: Isomerases • Unimolecularinterconversions(glucose-6-P  fructose-6-P) • Reactions usually almost exactly isoergic • Subcategories: • Racemases: alter stereospecificity such that the product is the enantiomer of the substrate • Mutases: shift a single functional group from one carbon to another (phosphoglucomutase) Recombinant II; Enzymes I

40. Category 6: Ligases • Catalyze joining of 2 substrates,e.g.L-glutamate + ATP + NH4+L-glutamine + ADP + Pi • Require input of energy from XTP (X=A,G) • Usually called synthetases(not synthases, which are lyases, category 4) • Typically the hydrolyzed phosphate is not incorporated into the product; it gets left behind as a free product Recombinant II; Enzymes I

41. Enzyme Kinetics • Kinetics: study of reaction rates and the ways that they depend on concentrations of substrates, products, inhibitors, catalysts, and other effectors. • Simple situation A B under influence of a catalyst E, at time t=0, [A] = A0, [B] = 0: • then the rate or velocity of the reaction is expressed as d[B]/dt. Recombinant II; Enzymes I

42. [B] Kinetics, continued t • In most situations more product will be produced per unit time if A0 is large than if it is small, and in fact the rate will be linear with the concentration at any given time: • d[B]/dt = v = k[A] • where v is the velocity of the reaction and k is a constant known as the forward rate constant. • Since [A] has dimensions of concentration and d[B]/dt has dimensions of concentration / time, the dimensions of k will be those of inverse time, e.g. sec-1. Recombinant II; Enzymes I

43. More complex cases • More complicated than this if >1 reactant involved or if a catalyst whose concentration influences the production of species B is present. • If >1 reactant required for making B, then usually the reaction will be linear in the concentration of the scarcest reactant and nearly independent of the concentration of the more plentiful reactants. • In fact, many enzymes operate by converting a second-order reaction into a pair of first-order reactions! Recombinant II; Enzymes I

44. Bimolecular reaction • If in the reactionA + D  Bthe initial concentrations of [A] and [D] are comparable, then the reaction rate will be linear in both [A] and [D]: • d[B]/dt = v = k[A][D] = k[A]1[D]1 • i.e. the reaction is first-order in both A and D, and it’s second-order overall Recombinant II; Enzymes I

45. Forward and backward • Rate of reverse reaction may not be the same as the rate at which the forward reaction occurs. • If the forward reaction rate of reaction 1 is designated as k1,the backward rate typically designated as k-1. Recombinant II; Enzymes I

46. Multi-step reactions • In complex reactions, we may need to keep track of rates in the forward and reverse directions of multiple reactions. • Thus in the conversion A  B  Cwe can write rate constantsk1, k-1, k2, and k-2as the rate constants associated with converting A to B, converting B to A, converting B to C, and converting C to B. Recombinant II; Enzymes I

47. [ES] Michaelis-Menten kinetics (G&G §13.2) t • A very common situation is one in which for some portion of the time in which a reaction is being monitored, the concentration of the enzyme-substrate complex is nearly constant. Thus in the general reaction • E + S  ES  E + P • where E is the enzyme, S is the substrate, ES is the enzyme-substrate complex (or "enzyme-intermediate complex"), and P is the product • We find that [ES] is nearly constant for a considerable stretch of time. Recombinant II; Enzymes I

48. Michaelis-Menten rates • Rate at which new ES molecules are being produced in the first forward reaction is equal to the rate at which ES molecules are being converted to (E and P) and (E and S). • Formation of ES is first-order in both [S] and available [E] • Therefore: rate of formation of ES from left =vf= k1([E]tot - [ES])[S]because the enzyme that is already substrate-bound is unavailable! Recombinant II; Enzymes I

49. Equating the rates • We started with the statement that the rate of formation of ES and the rate of destruction of it are equal • Rate of disappearance of ES on right and left isvd = k-1[ES] + k2[ES] = (k-1+ k2)[ES] • This rate of disappearance should be equal to the rate of appearance • Under these conditions vf = vd. Recombinant II; Enzymes I

50. Derivation, continued • Thus since vf = vd by assumption, k1([E]tot - [ES])[S] = (k-1+ k2)[ES] • Km (k-1+ k2)/k1 = ([E]tot - [ES])[S] / [ES] • [ES] = [E]tot [S] / (Km + [S]) • But the rate-limiting reaction is the formation of product: v0 = k2[ES] • Thus v0 = k2[E]tot [S] / (Km + [S]) Recombinant II; Enzymes I