5. From the genetic code to enzyme action

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1 Introductory biophysics A. Y From the genetic code to enzyme action Edoardo Milotti Dipartimento di Fisica, Università di Trieste

2 The structure of DNA Images from

3 ... initially stated in

4 This code is universal, it is the same for all living beings on earth, from viruses to complex organisms.

6 The chemical structure of nucleic acids phosphate sugar base (two purines, A and G + nucleotide two pyrimidines, C and T in DNA, C and U in RNA)

7 RNA is very similar to DNA, but it differs in three main ways: Unlike double-stranded DNA, RNA usually is a single-stranded molecule in many of its biological roles and has a much shorter chain of nucleotides. However, RNA can, by complementary base pairing, form intrastrand double helixes, as in trna. While DNA contains deoxyribose, RNA contains ribose. The additional hydroxyl group makes RNA less stable than DNA because it is much more prone to hydrolysis (t 1/ years for single bond hydrolysis in DNA; t 1/2 4 years for single bond hydrolysis in RNA). The complementary base to adenine is not thymine, as it is in DNA, but uracil.

8 Hairpin RNA structure (bent single RNA strand with intrastrand bonds)

9 Copying DNA into messenger RNA (mrna) RNA polymerase is a huge factory with many moving parts. It is composed of a dozen different proteins. Together, they form a machine that surrounds DNA strands, unwinds them, and builds an RNA strand based on the information held inside the DNA. Once the enzyme gets started, RNA polymerase marches confidently along the DNA, copying RNA strands thousands of nucleotides long. (adapted from PDB: molecule of the month,

12 mrna synthesis

The process of transcription can be visualized by electron microscopy; in fact, it was first observed using this method in 1970.

13 The process of transcription can be visualized by electron microscopy; in fact, it was first observed using this method in In these electron micrographs, the DNA molecules appear as "trunks," with many RNA "branches" extending out from them. When DNAse and RNAse (enzymes that degrade DNA and RNA, respectively) were added to the molecules, the application of DNAse eliminated the trunk structures, while the use of RNAse wiped out the branches. The RNA processing machinery is contained inside structures called "terminal knobs" in nascent RNA transcripts. Terminal knobs are visible in this electron micrograph of chromatin spreads from yeast. Dragon. F. et al. A large nucleolar U3 ribonucleoprotein required for 18S ribosomal RNA biogenesis. Nature 417,969 (2002).

19 About 95% of the multiexon genes undergo alternative splicing.

20 alpha-amanitin is a small polypeptide with 8 amino acids, it is found in deadly mushrooms of the Amanita genus like the death cap and it works its deadly action by inhibiting RNA- Polymerase II and III

The mrna is decoded by ribosomes, which synthesize proteins Ribosomes are composed of two subunits: a large subunit, shown on the right, and a small

21 The mrna is decoded by ribosomes, which synthesize proteins Ribosomes are composed of two subunits: a large subunit, shown on the right, and a small subunit, shown on the left. Both subunits are composed of long strands of RNA, shown here in orange and yellow, dotted with protein chains, shown in blue.

22 Subunits are often indicated with their speed of sedimentation in centrifuge. This is the small subunit 30S from Thermus thermophilus: proteins in blue, and the single ribosomal RNA chain in orange

23 This is the large subunit 50S from Haloarcula marismortui: proteins in blue, and the two ribosomal RNA chains in orange and yellow; the active site is shown in green (at the center)

25 from the slides of V. Ramakrishnan Nobel Prize lecture, dec.

27 from the Nobel Prize lecture of A. Yonath, dec. 2009

28 from the Nobel Prize lecture of V. Ramakrishnan, dec. 2009

30 from the slides of V. Ramakrishnan Nobel Prize lecture, dec. 2009

31 from the slides of V. Ramakrishnan Nobel Prize lecture, dec. 2009

32 from the slides of V. Ramakrishnan Nobel Prize lecture, dec. 2009

33 A few numbers on ribosomes: there are approximately 10 6 ribosomes/cell ( in liver cells) error rate about processing rate about 5 40 peptide bonds/s two units small: molecular weight about 0.85 Mdalton, RNA about 1600 nucleotides, 21 proteins large: molecular weight about 1.5 Mdalton, RNA about 3000 nucleotides, 34 proteins antibiotics often act against bacterial ribosomes (about 40% of all known antibiotics; molecular weight of antibiotics about dalton; many work by steric hindrance)

34 Transfer RNA (trna) selects the aminoacid that corresponds to a given codon and transports it to the ribosome

35 THE CRYSTAL STRUCTURE OF YEAST PHENYLALANINE TRNA AT 1.93 A RESOLUTION (from the Nucleic Acid Database

37 One end of a trna molecule has an anticodon three nucleotides that can form base pairs with a codon in mrna. At its other end, the trna carries an amino acid specified by the mrna codon. A trna that carries its specified amino acid is often called a charged trna. Each trna can bind one specific type of amino acid. Therefore, a cell must contain at least one trna for each of the 20 common amino acids. Enzymes known as trna transferases recognize the unique features of a trna and attach the correct amino acid. These enzymes are the key to the transfer of genetic data in protein synthesis. They read the language of the genetic code found at one end of a trna and match the code to a specific amino acid. (adapted from P. Jones, The Genetic Code, Chealsea House, 2011)

41 What do proteins do after synthesis? They have many roles, and one very important one is to act as catalysts. In this case they are called enzymes. Most enzymes are proteins. Some of them are specific RNA sequences (ribozymes, ribonucleic acid enzymes)

Consider the fact that cells are affected by oxidative stress.

electroncarrying riboflavin (vitamin B2) can encounter an oxygen molecule and

42 Consider the fact that cells are affected by oxidative stress. This is due to Reactive Oxygen Species, and it damages both DNA and other structures. Oxidative stress is part of the normal life of cells, for instance an electroncarrying riboflavin (vitamin B2) can encounter an oxygen molecule and convert it to the dioxide(1 ) anion O These dioxide anions are highly reactive and can damage other molecules, DNA in particular. How do cells defend themselves?

43 There are several important defense mechanisms against oxidative stress. One of them involves superoxide dismutase (SOD) which converts the dioxide(1 ) anion O 2- into oxygen (O 2 ) or hydrogen peroxide (H 2 O 2 ) Another one is catalase, which converts the still dangerous hydrogen peroxide into water and oxygen (O 2 ). Still another is peroxiredoxin which also converts hydrogen peroxide into water and oxygen (O 2 ).

44 The structure of catalase in human red blood cells: a protein with four polypeptide chains, each more than 500 aminoacids long.

45 Superoxide dismutase and catalase are enzymes, i.e., biological catalysts. Enzymes greatly accelerate specific chemical reactions. Enzymes are mostly proteins, however some of them are based on RNA (ribozymes). Our understanding of enzyme action is largely based on Transition State Theory (TST).

K eq = exp ΔG 0 RT Exponential dependence on DG 0 ( R 8.314 J K 1 mol 1, RT 2.

46 K eq = exp ΔG 0 RT Exponential dependence on DG 0 ( R J K 1 mol 1, RT 2.5 kj mol 300 K) this is close to the binding energy of hydrogen bonds in water 5 kcal/mole 21 kj/mole

47 Transition-state theory (TST) The idea is that a better description of chemical reactions can be obtained introducing a temporary, unstable state, the transition state, as in the following example of hydrogen bond exchange: H A H B + H C H A H B H C H A + H B H C transition state The l.h.s. of this chemical equation is assumed to be the limiting step of the reaction

49 The reaction coordinate In chemistry, a reaction coordinate is an abstract one-dimensional time-like coordinate which represents progress along a reaction pathway. It is usually a geometric parameter that changes during the conversion of one or more molecular entities, and can sometimes represent a real coordinate (such as bond length, bond angle...) In the formalism of transition-state theory the reaction coordinate is that coordinate which leads smoothly from the configuration of the reactants through that of the transition state to the configuration of the products. The reaction coordinate is typically chosen to follow the path along the gradient of potential energy from reactants to products.

50 A more general scheme... activation barrier

51 Now consider the reaction A + B K = X = k 0! P + Q the first step is fast and nearly at equilibrium for which we can write d[p ] dt = k 0 [X = ]=k[a][b] k has units [time] -1 [concentration] -1 k is a rate, units are [time] -1 and the equilibrium constant of the first step is K = = [X = ] [A][B] = k k 0 / exp G = RT

52 Since K = = [X = ] [A][B] = k k 0 / exp G = RT we find k / k 0 exp G = RT

53 Lets rewrite k' in a more physical way... k is a frequency and therefore hk is an energy; the only other energy scale in the breakdown of the transition state is the thermal energy k B T, and therefore we measure k' in units of k B T k k T B h k = κ k T B h Finally the rate at which the initial reactants are converted into the transition state is k = apple k BT h exp G = RT

54 Arrhenius plots k = apple k BT h exp G = RT apple = apple k BT S = h exp R H = = A exp RT = apple k BT h exp exp H = RT H = T S = RT ln k =lna H = RT this term is only weakly (logarithmically) dependent on temperature Arrhenius plot for the uncatalyzed hydrolysis of a- and b-1-methylglucopyranosides

55 <latexit sha1_base64="0stuhb8ktklsnafetiazyp0u2lc=">aaacmhicbzbns8mwgmctx+d82/tgwutwcj5gk4jehkexjxodcl0zazpqwnqu5kkwsj+nv734afqkxv0upl0ptn0g8of/voyxpfiycjx3pde/sli03fhprq6tb2y22ls3rmwa8qftuum7gbourcihiedyu1rzgges3wbj8zj/+8i1esq5hknk/zjejyi Non-specific, temperature dependent acceleration d[p ] dt = k[s] From A. Radzicka and R. Wolfenden, Science 267 (1995) 90

56 Enzymes Enzymes are biological catalysts which share several important properties they lead to much higher reaction rates for catalyzed processes (acceleration factors in the range ) they make reactions happen under mild conditions they are very specific the catalysis can be controlled and tuned In the context of TST, enzymes work by lowering the activation barrier.

58 Since k = apple k BT h exp G = RT if an enzyme lowers the energy barrier by that the new value of the rate constant is G =, we see k Enz = apple k BT h exp G = G = RT = k exp G = RT This is a very sensitive function of the barrier height and enzymes achieve large acceleration factors with small energy changes.

59 from R. Wolfenden and M. J. Snider, The Depth of Chemical Time and the Power of Enzymes as Catalysts, Acc. Chem. Res. 34 (2001)

60 Michaelis-Menten kinetics E + S k + ES k cat E + P k v = d [ P ] dt d[ S] dt d[ E] dt d[ ES] dt = k cat [ ES] = k + [ E] [ S]+ k [ ES]+ v S = k + [ E] [ S]+ k [ ES]+ k cat [ ES] = k + [ E] [ S] k [ ES] k cat [ ES] = d [ E ] dt

61 We must add the equation for enzyme mass conservation [ E] 0 = [ E]+ [ ES] When the system is in quasi-stationary conditions, we obtain k [ ES]+ k cat [ ES] = k + [ E] [ S] = k + [ E] 0 [ ES] [ ] ( ) S then, using ES = v k cat we find v k cat = ES [ ] = [ E] 0 S k + k cat k + [ ] + [ S] v = k cat E k + k cat k + [ ] 0 [ S] + [ S] = V S max [ ] K m + [ S]

62 [ ] [ ] v = V S max K m + S Michaelis-Menten equation where we let V max = k cat [ E] 0 K m = k + k cat k + maximal velocity Michaelis constant

63 VêVmax

64 Notice that at small concentrations v = V max S K m + S [ ] [ ] = k cat [ E] 0 [ S] K m + [ S] k cat K m approximation for small substrate concentration [ E] 0 [ S] and therefore the ratio k cat /K m can be interpreted as the normal rate of a binary reaction. But k cat K m [ E] 0 can also be interpreted as the rate of an elementary unary reaction, and therefore the ratio k cat /K m represents the acceleration factor per enzyme molecule of the spontaneous conversion of the substrate.

66 From A. Radzicka and R. Wolfenden, Science 267 (1995) 90

67 Lock-and-key specificity

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