Chapter 36: Complex Reaction Mechanisms Problem numbers in italics indicate that the solution is included in the Student's Solutions Manual. Questions on Concepts Q36.1) How is a simple reaction different from a complex reaction? A simple reaction is any reaction that consists of the single reaction step. A complex reaction is any reaction that occurs in two or more elementary steps. Q36.2) For a reaction mechanism to be considered correct, what property must it demonstrate? In order to be valid, a proposed reaction mechanism must agree with the experimentally determined rate law. Q36.3) What is a reaction intermediate? Can an intermediate be present in the rate law expression for the overall reaction? A reaction intermediate is a chemical species that is formed during the course of a reaction, but decays as the reaction proceeds resulting in product formation, ‘The reaction intermediates must be consumed prior to the completion of the reaction, therefore reaction intermediates will not appear in the overall reaction expression. Q36.4) What is the pre-equilibrium approximation, and under what conditions is it considered valid? ‘The pre-equilibrium approximation is somewhat related to the steady state approximation. ‘The pre- equilibrium approximation assumes a complex reaction of the form A+BE I+ 5P The principle assumption of the pre-equitibrium approximation js that equilibrium is between reactants A, B and the intermediate species I. 1 then reacts to form the produet P. This approximation is valid when equilibrium is established in advance of product formation, or when ky and ky >> ky. Q36,5} What is the one main assumption in the Lindemann mechanism for unimolecular reaction: ‘The principle assumption of the Lindemann reaction is that an activated complex is formed during the course of the reaction. This activated complex can then deactivate resulting in reformation of the reactant, or it ean decompose resulting in product formation, This activated complex assumption allows for the decomposition to be a linear function of the concentration of the activated reactant; therefore, the Lindemann mechanism is consistent with the experimentally determined animolecularity of the react 884Chapter 36/Complex Reaction Mechanisms Q36.6) How is a catalyst defined, and how does such a species increase the reaction rate? A catalyst is a chemical species that increases the rate of a chemical reaction without being consumed during the reaction. ‘The catalyst serves to set up an additional reaction coordinate for which the activation energy is reduced relative to the uncatalyzed reaction. Q36.7) What is an enzyme? What is the general mechanism describing enzyme catalysis? An enzyme is essentially a biological catalyst. The “lock and key” model of enzyme activity is discussed in the text where the catalyst binds a substrate in a very specific location and orientation. ‘Once the enzyme-substrate complex is formed, the product is produced and free enzyme is regenerated. Q36.8) What is the Michaelis-Menten rate law? What is the maximum reaction rate predicated by this rate law? The Michaelis-Menten mechanism that describes enzyme catalysis is as follows: 4 E+ SS 2ES—E +P where E is the enzyme, S is the substrate, ES is the enzyme-substrate complex and P is the product. The Michaelis-Menten rate law is derived based on the assumption that the initial concentration of the substrate ([SJo) is much greater than the initial concentration of the enzyme ([EJo). The rate law is expressed as KISHEE], (S|, +K,, where Ky is the Michaelis constant, which combines the rate constants, Q36.9) How is the standard enzyme kinetic scheme modified to incorporate competitive inhibition? What plot is used to establish competitive inhibition and to determine the kinetic parameters associated with inhibitio A competitive inhibitor is a molecule that structurally resembles the substrate of interest, but does not react. A competitive inhibitor binds to the active site of the enzyme. The kinetics are changed in that a third reaction step must be considered, the equilibrium of the docking of the inhibitor, expressed as Evel The consequence of competitive inhibition is a decreased reaction rate. As with the Michaelis-Menten kinetics, a modified Lineweaver-Burk plot is employed. (Q36.10) What is the difference between a homogeneous and a heterogeneous catalyst? For a homogeneous catalysis both the catalyst and the substrate exist in the same phase. Heterogeneous catalysis occurs when the catalyst and the substrate molecules exist in different phases.Chapter 36/Complex Reaction Mechanisms Q36.11) What are the inherent assumptions in the Langmuir model of surface adsorption? The Langmuir model arises from three assumptions, (1) Adsorption finishes once one monolayer of coverage results (2) The surface is uniform and all adsorption sites are equivalent. (3) The occupaney of a site will not affect the adsorption or desorption processes in adjacent sites. Q36.12) What is a radical? What elementary steps are involved in a reaction mechanism invol radicals? Radicals are chemical species that contain an unpaired electron. The general mechanism for rad reactions involves three steps: (1) Initiation—the radical species is formed from a precursor. (2) Propagation—one radical species generates a second radical species, generally by abstraction of an atom or larger moiety. (3) Termination—the radical species eventually recombines with a second radical to form a non-radical species. Q36.13) In what w. are radical polymerization reactions similar to radical reactions in general? The radical polymerizations are very similar to the general radical reaction mechanisms in that they involve initiation, propagation, and termination steps. 36.14) What is photochemistry? How does one calculate the energy of a photon’ Photochemistry is the initiation of a chemical reaction through the absorption of energy in the form of photon. The photon is a quantum particle of light with energy: Erion where / is Planck’s constant and vis the frequency of the light Q36.15) What depopulation pathways oc triplet state? from the first excited singlet state? For the first excited There are three main pathways for decay of the first excited singlet state: emitting a photon or fluorescence, non-radiative decay or internal conversion, and intersystem crossing to the triplet state. The first excited triplet state decays by emitting a photon or phosphorescence, and non-radiative decay or internal conversion. excited state lifetis na Stern, Q36.16) What is the expected variation i Volmer plot? ie with quencher concentration is ‘The fluorescence lifetime is inversely proportional to the concentration of the quenching species. As the quencher concentration increases, the fluorescence lifetime decreases. The slope of the plot is the rate constant for quenching 886Chapter 36/Complex Reaction Mechanisms Q36.17) What is the distance dependence for fluorescence resonance energy transfer predicted by the Forster model? ‘The efficiency of energy transfer is dependent on distance as follows: Lif nee Q36.18) What two factors influence the electron transfer rate constant according to Marcus theory? In Marcus theory the rate of electron transfer is dependent on the difference in Gibbs energy between the product and reactant (AG), and the reorganization energy accompanying electron transfer (2) as follows: _(4Gr+ ay ak with AGT Problems P36.1) A proposed mechanism for the formation of N)Os from NO> and Osis, NO, + 0, 4NO, +0, NO, +NO, +M—4->N,0, +M Determine the rate law expression for the production of Nos given this mechanism, AlN.04] 2 [NOs][NO,] INO.) ; [NO, ][0s]-,[NO, ][NO, ] 887Chapter 36/Complex Reaction Mechanisms Applying the steady state approximation to the intermediate NOs and substituting back into the differential rate expression for NzOs yields: d{NO. J di k,[NO,][NO,]= & [NO,][0,] 410] =k [NO,][0,]-&[NO, ][NO,] [No,]= k d{N.0.] 5 W011 4, [N0,J[N0,] =41N0,][ #103} =k [NO, ][0,] The mechastism predicts that the reaction is first order in NO» and Os, second order overall. 36.2) The Rice-Herzfeld mechanism for the thermal decomposition of acetaldehyde (CH3CO) is CH,CHO—*>CH, -+CHO- CH, -+CH,CHO—! >CH, +CH,CHO CH,CHO-—*+CO+CH,- CH, -+CH,-—*>C,H, ine the rate of methane (CHs) formation, Using the steady-state approximation, deter ‘The differential rate expressions for methane and relevant intermediate species are: a[cH,]_ a a[CH,CHO] (cn, J[cu,cuoj {cH, ][cH,CHO]-&,[CH,CHO-] ; [CH,CHO] - &, [CH, -][CH,CHO]+&, [CH,CHO ]- 2k, [CH, } Applying the steady state approximation to the differential rate expressions for the intermediates: d[CH,CHO-] act] 4 dt dt 888.Chapter 36/Complex Reaction Mechanisms or 0 =k, [CH, -][CH,CHO}- &,[CH,CHO-] =k, [CH,CHO] - &, [CH, -]CH,CHO] +, [CH,CHO-]-2k, [CH, | Adding the above two equations yields the following expression: 2k, [CH, -] = &, [CH,CHO] ki, 2k, [cH, H,CHO]” Substitution into the rate expression for [CH] yields a[cu,] dt acu, =4 sz-lcr.cHo} "Jessen =k, [CH,CHO}” where k, P36.3) Consider the following mechanism for ozone thermal decomposition: 0, 20,+0 0, +0—* 520, a) Derive the rate law expression for the loss of Os 'b) Under what conditions will the rate law expression for Os decomposition be first order with respect to Os? a) MO a fojee [0,][0]-&, [0;][0] b) Atomic oxygen is the intermediate species, and the differential rate expres species is: A214 (0,)-& [o,Jf0]-H[0,][0] Applying the steady-state approximation to the differential rate expression for O yields: 889,Chapter 36/Complex Reaction Mechanisms Ape -slOs}-+f0sl0}-A(0, 0} 4 [0,}=# [0,]f0}+4[0, 10] 410) toy k,[0,]+k,[0,] Substituting this result into the differential rate expression for ozone yields: ae =-k[0,]}+& [0,][0]-&,[0,][0] = 40.) +{0](& [0.]-6[0,)) --s1o.}e[ Af gg }l0.1-st0.) -k,[0,]+&,[0, ae toll iohEb}) po. EaLOelAelOs], “Ai lO-]+4 [04] Aol; "[O.}r&[0,]"& eee) 2k [O° [0,}+4.[0] iF b,[0,]>> k,[0,] 36.4) The hydrogen-bromine reaction corresponds to the production of HBr from Hp and Br) as follows: H, + Br, —+2HBr. This reaction is famous for its complex rate law, determined by Bodenstein and Lind in 1906: {HBr} _&[H, [Br] dt m[HBr] Bs] id mare constants. It took 13 years for the correct mechanism of this reaction to be proposed, and this feat was accomplished simultaneously by Christiansen, Herzfeld, and Polyani. The mechanism is as follows:Chapter 36/Complex Reaction Mechanisms Br —22Br Br-+H, —2>HBr+H- H+ Br, > HBr+ Br: HBr+H-—“4H, +Br- Construct the rate law expression for the hydrogen—bromine reaction by performing the following steps: a) Write down the differential rate expression for [HBr] +b) Write down the differential rate expressions for [Br] and [H]. ) Because Br and H are reaction intermediates, apply the steady-state approximation to the result of part (b). 4) Add the two equations from part (c) to determine [Br] in terms of [Bro] €) Substitute the expression for [Br] back into the equation for [H] derived in part (c) and solve for [H]. £) Substitute the expressions for [Br] and [H] determined in part (e) into the differential rate expression for [HBr] to derive the rate aw expression for the reaction. o) MPT y pe jf}, fe [Bs ]-e [860] b) ME) A e]-24 [Br] ~&, [Br-][H,]+& [H-][Br, J+é,(HBr][H ] ay BD 4, (oe, [H Jf] eer] ©) Applying the steady state approximation to [Br-] and [H-] and adding yields 2 0= 2k, [Br, ]- 2k, [Br-] —& [Br-][H,]+ 4, [H-][Br, ]+ &, [HBr][H-] Ok fe fH, ]-4 [FEB] A, [64] (4 ] d 0=2k,[Br,]-2k ,[Br-]) Ee &,[Br-][H,]=4,[H-][Bn ]+4, [HBr][H ] &,[Br-][H, Besa tH] [Br e) Ba Y*[H,] k,(Br,]+, [HBr] “ey 891Chapter 36/Complex Reaction Mechanisms 1) Now, substitution into (a) yields {HBr} dt (orlft) +4 LB], [ABELL] ipa tne) ka, /E tos) k [HBr] _&,[Br,]+&, [HBr Ba P(t] feel) Tote we mh Be] (ed * [Bee [Br] ‘ [Bn )[H JfH] k,[Br, ]+, [HBr] [Be )*[H,] 36.5) a) For the hydrogen-bromine reaction presented in Problem P36.4, imagine initiating the reaction with only Br and Hz present. Demonstrate that the rate law expression at ¢ = 0 reduces to (42) =o (4) oot” dt b) The activation energies for the rate constants are as follows: Rate Constant AE, (ki/mol) ky 192) ke 0 ks 74 What is the overall activation energy for this reaction? ¢) How much will the rate of the reaction change if the temperature is increased to 400. K from 298 K? 892Chapter 36/Complex Reaction Mechanisms d) a) Att=0 0 [Br] =[BroJo [Hz] = {Halo [HBr] = ‘Thus: (4) _*[Be | [H), ol? tu. =#{o5]2 [01], a io 61 +m. ° [Bs], sl (EP) =a Fest to realy (PY Y4 ost *-n4 ma) t2a(4) e wr Yosh t-te) } fi esse! tra,(24) em Therefore, Ey = 59 kJ mol se fie 00) FALE yoo ©) 434 Si mat TALE ype 2 Um er rs) P36.6) For the reaction I" (aq)+ OCI (aq)7—* OF (aq) + CF (aq) occurring in aqueous solution, the following mechanism has been proposed: Cl +H,0—> HOCI+OH- TF +HOCI—2 + HOL+Cl HOL+OH —*+H,0+01- a) Derive the rate law expression for this reaction based on this mechanism. (Hint: [OH] should appear in the rate law.) 893Chapter 36/Complex Reaction Mechanisms b) The initial rate of reaction was studied as a function of concentration by Chia and Connick [J. Phys. Chem. 63 (1959), 1518}, and the following data were obtained: [hay [OCT o(M) | [OH js (wt) Initial Rate (M 20x10° 15x 10% 1.00 1.8 x10" 4.0 x10° 1.5 x 10% 1.00 3.6107 20x10 3.0.x 10% 2.00 18x 10% 4.0 x 10% 3.0 x 107 1.00 72x10 Is the predicted rate law expression derived from the mechanism consistent with these data? a) {or J. k, [HOI], OH} ‘The intermediate species have rate expressions (with steady state approximation) aioe} =k [ocr |[H,0]~k ,[Hoct]] OH }—x, [1 ][Hoct] avo) =k, [IF ][Hoci]-& [Hor[ oH ]=0 Solving the last expression for [HOI] and substituting into the differential rate expression for [OF] yields: afor} dt _[F ][Hocl] Next, [HOCI] must be expressed in (erms of reaetants and [OH ]. Rearranging the steady-state approximation applied to the differential rate expression for [HOCI] results in the following: [ocr ][#,0] )[OH ] +4 [1 ] This result is substituted into the differential rate expression for [OI] to yield: a[or] kk [oct ][H.0][F ] dt «fo Je [] [Hoct Inspection of the concentrations employed in the table suggests that ko[1"] << k,[OF | resulting in: afor ] a &fo[T J[ocr ][H,0} [ou] 804Chapter 36/Complex Reaction Mechanisms. A markedly similar expression is obtained using the pre-equilibrium approximation to determine {HOCI} b) Consider set 1 & 2: [I] is doubled, doubling the rate set | & 3: Doubling [OCI] and [OH results in no net change in rate set 1 & 4: Doubling [I] and [OCI"] quadruples the rate. These results confirm the rate expression. P36.7) Using the pre-equilibrium approximation, derive the predicted rate law expression for the following mechanism: A, 2A AVB—25P ‘The rate expression for the formation of the product is APN tage dt The equilibrium constant can be expressed as therefore, Substituting this into the rate expression for P yields: SPA) ta.r°ta) APL TATPEBL where ky P36.8) Consider the following mechanism, which results in the formation of product P: ARS BEC BP 895Chapter 36/Complex Reaction Mechanisms Ifonly the species A is present at r= 0, what is the expression for the concentration of P as a function of time? You can apply the pre-equilibrium approximation in deriving your answer. Using the pre-equilibrium approximation, we can express [B] and [C] in terms of [A] as follows: (I= FAl= 404) [c]= API (5-44) ~[B]=K,[B]= Kk, [A] Next, from mass conservation: (4), =[4}[B}+(C]+[P] HAL 9 AA), a8) aC] al?) at dd tt afr] -(“4. @[B) , 1) dt dd dt afe)__ fafa], a(x [A]) MELA) dat dt at a[a] =-(1+K,+K,K,)FF (4K Rk) Setting the two differential rate expressions for [P] and integrating yields: (14K, + xx Hla, [a] { W dla, tx j [a] “lis +K,K,) ' Ifa pre-equilibrium is established rapidly before any product formation: [A], =[Ah" +[B]"+1ch" = (14K, +K,K,)[A]" 896Chapter 36/Complex Reaction Mechanisms [P}-[A], “(4B [e) =[4],-(A}+ fA] +4. [A)) = [4], “(0+ KKK )AT — “ky -ta)-2%o P36.9) Consider the gas-phase isomerization of cyclopropane. Are the following data of the observed rate constant as a function of cyclopropane pressure consistent with the Lindemann mechanism? P (Tor) kQ0*s") [| P (Torr) kd0"s') 84.1 2.98 137 1.30 34.0 2.82 0.569 0.857 110 2.23 0.170 0.486 6.07 2.00 0.120 0.392 2.89 154 0.067 0.303 If the data obeyed the Lindemann mechanism, then a plot of the data would fit the equation tok tt fm ik [M] . . . 1 Therefore, the Lindemann mechanism predicts that a plot of + versus 4 should bea straight line. This plot is provided below, and demonstrates that the plot shows significant curvature so that the Lindemann mechanism does not provide an adequate description of the reaction mechanism. 897Chapter 36/Complex Reaction Mechanisms as “+ 2s & Sas os ° 0 2 «© 6 8 6 2 ww Ps (torr!) P36.10) In the discussion of the Lindemann mechanism, it was assumed that the rate of activation by collision with another reactant molecule, A, was the same as collision with a non-reactant molecule, M, such as a buffer gas. What if the rates of activation for these two processes are different? In this case, the mechanism becomes A+M a= AttM ATA SAMA At 4 5P a) Demonstrate that the rate law expression for this mechanism is &,(« LAl[M]+4, (AF) ~ ky[M]+e, [A] +k, b) Does this rate law reduce to the expected form when [M] = 0? a[P] =k fA") a LA) AAT [alge (0 LAT LM ABA] a) Applying the steady state theory to A* yields [ae] +e [Ar]IM] +4 [A114] = 4 [4] M}+4 [4] 898Chapter 36/Complex Reaction Mechanisms 9 ALAM] +4 [a] IS? [Mee alrs Substituting back into the differential rate expression for the produet yields: apy _&(4LAIIM]+4,[4F) dk [M}+k [A], b) If the concentration of M goes to 0, then &(k kA[A] +h which is the Lindemann rate expression. P36.11) In the unimolecular isomerization of cyclobutane to butylene, the following values for kyy: a8 a function of cyclobutane pressure were measured: Py (Tor) 110 210 390 760 Kuni (S) 9.58 10.3 10.8 Wt Assuming that the Lindemann mechanism accurately describes this reaction, determine k, and the ratio Kalk. ‘The Lindemann mechanism predicts the following rel constant for the reaction and the concentration of reactant: ship between the unimolecular rate 399Chapter 36/Complex Reaction Mechanisms Therefore, a plot of kui ' versus P™ should yield a straight line, as shown below: y= 1893 + 0.0878 1.834 s torr k, P Thus, k; can be related to the slope kr (0.0821 L atm mol ' K')(350. K) ~ slope Tatm (1.8345 wom (8 mm k, &,=1.19x10' M's! ky. . ‘The ratio [is related to the intercept fi £1 (y-in\(h)=(0 0879 s)(1.19«10' M's") .05x10° M* hy 36,12) The enzyme fumarase catalyzes the hydrolysis of fumarate: Fumarate ++ 1,0 —> L-malate 900Chapter 36/Complex Reaction Mechanisms ‘The turnover number for this enzyme is 2.5 x 10° s ', and the Michaelis constant is 4.2 x 10° M. What is the rate of fumarate conversion if the initial enzyme concentration is 1 x 10 M and the fumarate concentration is 2 x 10 M? Recognizing that ky is the turnover number, the values provided in the problem can be used directly in the rate expression to determine the initial rate of reaction: k= 2.5 x 10° 8", Km = 4.2 x 10° M, [EJo= 1 x 10M, [S]o=2 x 104M. [SLL], k, rate, [S], + Ka (25x10 s")(2x104 M)(1x10% M) “(2x10 M)+(42x10°M) =2.45x10° Ms! P36.13) The enzyme catalase catalyzes the decomposition of hydrogen peroxide. The following data are obtained regarding the rate of reaction as a function of substrate concentration: [H2O>}o (M) 0.001 0,002 0.005 Initial Rate (Ms) [1.38 107 2.67 x 10) 6.00 x 10° ‘The concentration of catalase is 3.5 x 10° M. Use these data to determine ratema, Kw, and the turnover number for this enzyme, Analyzing the data using the Lineweaver-Burk plot: 1 iy rate, rate, rate, 901Chapter 36/Complex Reaction Mechanisms ‘The corresponding plot of ratey' versus [H0.] ' is as follows: 0099774 26557, Best fitto the data by a straight line yields the following equation: 1 1 —— =(0,698 s) —-+26.6M' rae, (8S) Tt . The ratema: is related to the intercept 1 1 y-int 266M =3.75x10? Ms! Plea. = Fle, With ratema, the Michaelis constant is determined using the slope: slope)x (rate... ) = (0.698 s}x(3.75x107 Ms! 2.63%10? M Finally, the turnover number is determined using rafénas and [Ep as follows: rates, = ky[E], rate, 1.0810" s 902Chapter 36/Complex Reaction Mechanisms. 36.14) Peptide bond hydrolysis is performed by a family of enzymes known as serine proteases. The name is derived from a highly conserved serine residue in these enzymes that is critical for enzyme function. One member of this enzyme class is chymotrypsin which preferentially cleaves proteins at residue sites with hydrophobic side chains such as phenylalanine, leucine, and tyrosine. For example, N- benzoyl-tyrosylamide (NBT) and N-acetyl-tyrosylamide (NAT) are cleaved by chymotrypsin. a.The cleavage of NBT by chymotrypsin was studied and the following reaction rates were measured as a function of substrate concentration: [NBT] (mM) | 1.00 2.00 4.00 6.00 8.00 Rate, (mM s") [0.040 0,062 [0.082 0.099 0.107 Use these data to determine Kiy and Rafemax for chymotrypsin with NBT as the substrate reaction rates versus substrate concentration b.The cleavage of NAT is also studied and the folloy were measured: [NAT] (mM) [1.00 2,00. 4.00 6.00 8.00 Rate, (Mm s') [0.004 | 0.008 0.016 0.022, 0.028) Use these data to determine Ky, and rafemar for chymotrypsin with NAT as the substrate. ) Constructing a Lineweaver-Bruk plot of I/rateg versus 1/[S]p should result in a straight line with slope equal to Ky/ratema:and y-intercept equal to ratemax: 1 Tale, TAleyy |S], Ale gy, YR ITT13x# 7.3213 o 02 ost Sk" (mv) 903Chapter 36/Complex Reaction Mechanisms Best fit by a straight line yields a y-intercept of 7.32 mM" s, the inverse of which is ratemax: oy 20-137 mM (7.32 mM" s) Plea, The best fit also yields a slope of 17.7 s. The product of the slope and ratemax is equal to Ky: K,, =(17.7s)(0.137 mM s')=2.42 mM constructed from the data: ) Repeating the analysis performed in part a, a Lineweaver-Burk plot 300 y= 245 82x + 3.3852 ° 02 oa os 08 1 12 {S]," (mM) Best fit by a straight line yields a y-intercept of 3.36 mM s, the inverse of which is rate ma Paley. —— __ 0.208 ms! (3.36 mM" s) The best fit also yields a slope of 245.8 s. The product of the slope and ratémay is equal to Ky: K,, = (245.8 s)(0.298 mM s)=73.3 mM P36.15) Protein tyrosine phosphatases (PTPases) are a general class of enzymes that are involved in a variety of disease processes including diabetes and obesity. In a study by Z.-Y. Zhang and coworkers [J. Medicinal Chemistry 43 (2000), 146], computational techniques were used to identify potential competitive inhibitors of a specific PTPase known as PTPIB. The reaction rate was determined in the presence and absence of inhibitor, I, and revealed the following initial reaction rates as a function of substrate concentration: 904Chapter 36/Complex Reaction Mechanisms. [S] (aM) Rateo (uM $"), Ratey (4M 8), (=o [I] =200 uM 0.299 0.071 0.018 0.500. 0.100, 0.030 0.820 0.143 0.042 1.22 0.250, 0.070 1.75 0.286 0.105 2.85 0.333 0.159 5.00 0.400 0.200 | 5.88 0.500 0.250 a) Determine Ky and ratejax for PTP1B. b) Demonstrate that the inhibition is competitive, and determine K,. a) Performing a Lineweaver-Burk plot for the uninhibited reaction yields: y= 3.9836 + 1.587 as 25 2 {Sr uM") Best fit to the data by a straight line yields the following equation: l =3.88s ah 1.ssx10° M's rate, is. 905Chapter 36/Complex Reaction Mechanisms ‘The maximum rate is equal to the inverse of the y-intercept: 1 1 Paley = int raleg., = 6.45%10" Ms! With ratemax. the Michaelis constant is determined from the slope: K,, =(slope )x (rate, ) = (3.88 s)x(6.45x10" Ms") K,, =2.5x10°M b) With the inhibitor present, the modified Lineweaver-Burk equation is used where: I ry KL Tale, Tiley. Paley, [5], Again, a plot of the inverse of the initial rate with the inverse of substrate concentration should yield a straight line. The plot is as follows: y= 16.34% + 1.2817 so 0 0s tts 238k 2 [sh aM") Best fit to the data by a straight line yields the following equation: u 63 s 1 1.28%10° M's rate, is. 906Chapter 36/Complex Reaction Mechanisms ‘The maximum rate is given by the inverse of the y-intercept: 1 1.28%10° M's raley.. =7.94x10" Ms" Paley, With ratema, and the slope of the line the apparent Michaelis constant is: slope)x (rate...) =(16.31 8)x(7.94x107 Ms‘) =13x10°M With Ky, Kn’ and [I], Kyis determined as follows: f K my K, 20010 M 130M) 2.5x10° M =4.76x10° M K, P36.16) The rate of reaction can be determined by measuring the change in optical rotation of the sample as a funetion of time if a reactant or product is chiral. This technique is especially useful for kinetic studies of enzyme catalysis involving sugars. For example, the enzyme invertase catalyzes the hydrolysis of sucrose, an optically active sugar. The initial reaction rates as a function of sucrose concentration are as follows: | [Sucrose]o (M) Rate (Ms") 0,029 0.182 0,059 0.266 0.088 0.310 0.117 0.330 0.175 0372 0.234 0.371 Use these data to determine the Michaelis constant for invertase. 907Chapter 36/Complex Reaction Mechanisms ‘The Lineweaver—Burk plot and best fit to the data is as follows: y= 0.0947% + 2.1979 ° 5 wo SSW {s}' (M") ‘The equation for the best-fit line is: 1 = 0.0947 5 1 -+2,1979 M's rate, {s], ‘The maximum rate is equal to the inverse of the y-intercept 1 y-int 2.20M's Paleo, = 0-455 MS PAle gg. = ‘The Michaelis constant is equal to the product of rafénax and the slope: K,, = (slope) x( rate...) = (0.0947 s) (0.455 Ms"') K,, =0.0431M P36.17) The enzyme glycogen synthase kinase (GSK-3/ ) plays a central role in Alzheimer’s disease. The onset of Alzheimer’s disease is accompanied by the production of highly phosphorylated forms of a protein referred to as“ .” GSK-3f contributes to the hyperphosphorylation of ¢ such that inhibiting the activity of this enzyme represents a pathway for the development of an Alzheimer’s drug. A compound known as Ro 31-8220 is a competitive inhibitor of GSK-3/ . The following data were obtained for the rate of GSK-3f activity in the presence and absence of Ro 31-8220 [A. Martinez et al. J. Medicinal Chemistry 45 (2002), 1292}: 908Chapter 36/Complex Reaction Mechanisms [S1GiM) [Rate (uM ,[1]=0 [Rateo (uM s“) 1] =200 uM 66.7, 4.17 «10% 3.33 x10" 40.0 3.97 x 10% 2.98 x 10* 20.0 3.62 x 10% 2.38 x10" 13.3 3.27x10* 1.81% 10* 10.0 2.98 x 10* 1.39x10* 6.67 2.31 x 10% 1,04 x 10* Determine Kyy and rates for GSK-3f and, using the data with the inhibitor, determine K,,* and K,. Analyzing the data without inhibitor using a Lineweaver~Burk plot yields: 0900000 90000 ¥ =. 296900 + 2.116407 . 40000000 30000000 < 28000000 20000000, rate" (uM s) 1s000000 100000 © 002 008 008 pao? os 0G {Ss} iM") The best-fit straight line to the data yields the following equation: 1139x108 s 1 42.11<10% uM" s rate, [s], ‘The maximum rate is equal to the inverse of the y-intercept: 1 1 y 211x107 oM's =4.74x10" uM s" rate, rate, 909Chapter 36/Complex Reaction Mechanisms. the maximum rate and slope of the best-fit line, the Michaelis constant obtained as follows: = (1.39% 108 s (4.7410 jiM s) Using the inhibited data, the Lineweaver-Burk plot is: 120000000 ‘vegooe000 | ¥ » S.O1E+08x + 2.026407 seoo0000 0000000 rate” (uM" s} 0000000 20000000 © 002 0.08 0.06 008 01 Oz ae 916 [s}' (aM) Best fit by a straight line to the data yields the following equation: 1 1 7 1, 5.01x10° s + 2.01x107 pM 's. rate, “Ts, n ‘The maximum rate with inhibitor is equal to the inverse of the y-intercept pr 2.01510" aM s =4.98x10* uM‘ s ‘The apparent Michaelis constant is given by: K1, =(slope) (rate...) =(3.01x10° s)>{4.98x10* M's) K. =24.9 uM 910Chapter 36/Complex Reaction Mechanisms Finally, the K; value is given by: 200uM a ( 4.9. at) kK, 6.49 uM = 70.4 uM 36.18) In the Michaelis-Menten mechanism, it is assumed that the formation of product from the enzyme-substrate complex is irreversible. However, consider the following modified version in which the product formation step is reversible: E+ SS ESE +P. Derive the expression for the Michaelis constant for this mechanism in the limit where [Slo >> [E]o. The reaction rate in terms of the differential rate expression for product formation is AP, fe5)-1, (810) Using the steady state for [ES] af 0=k,[E][S]-(« +4, )[ES]+&,[E][P] dt Now, noting that (EJ. = [E] + [ES], and [S]o ourselves to early times so that [ES] and [P] are small and [S equation, [ES} is given by: 0 =k {E], [S], ~& [ES][S], (e+ )[ES]+ 4 [E], [P]-42[ES]P] LIS], +*.1E)(P] J +* 2fP]+(K, +4) [S] + [ES] + [P]. As in the text, we confine |S]. Using the preceding If[S] >> [P], [EJo, we can neglect the term involving [E]o[P] in the preceding equation, onChapter 36/Complex Reaction Mechanisms Pl tes «[E][P] “antag *Mlek teste 4h {E],[S] e “EBD -G Pte ek) SI] _AALELIS], eh _ALEL AS} kal AISLE +h) «teh a IP'the term involving [P] i original ignored in the denominator of the preceding equation, then the aclis expression is recovered, P36.19) Reciprocal plots provide a relatively straightforward way to determine if an enzyme demonstrates Michaelis-Menten kinetics and to determine the corresponding kinetic parameters. However, the slope determined from these plots can require significant extrapolation to regions corresponding to low substrate concentrations. An alternative to the reciprocal plot is the Eadie-Hofstee plot where the reaction rate is plotted versus the rate divided by the substrate concentration and the data are fit to a straight line. a, Beginning with the general expression for the reaction rate given by the Michaelis-Menten mechanism: rearrange this equation to construct the following expression which is the basis for the Eadie~ Hofstee plot: CR) BB 912Chapter 36/Complex Reaction Mechanisms b. Using an Eadie-Hofstee plot, determine Rnas and Ky for hydrolysis of sugar by the enzyme invertase using the following data: [Suerose]o (M) Rate (Ms") 0.029 0.182 0) 0.266 0.088 0.310 0.117 0.330 0.175 _ 0.362 0.234 0.361 a) Rua [8 na Bol [S], +n Ry([S], + K.)= Row [S], Ryl[S], = Row [S],— RK R, R= Raa Ky (4) AUS, a) Using the data provided, a plot of rateg versus rateg/[S]p is constructed: 0.4 = y= -0.0392x+.0.4372 0.35 ty R= 0.9777 03 mk. 0.25 / 045, rate, (uM s°) o ° 0.05 o 1 2 3 4 5 6 7 rate {IS}, (8°) Best fit to a straight line yields a y-intercept of 0.437 M s', which is equal to Ratéma. The slope of the line is equal to -0.0392 M, which is equal to —K,, such that Ky, = 0.0392 M. 913Chapter 36/Complex Reaction Mechanisms ‘P36.20) Determine the predicted rate law expression for the following radical-chain reaction: A,—92A ABC A+B-—5P A-+P—49B ‘The differential rate of P formation is API a fay] -K LATE The rate expression for A- and B: are MA aa fa,)-b (41-6101 MIE] MET a Ay ABE RIAIE Applying the steady-state approximation for [B+] KAT] «(ATP ]= 614] +k,[P] [B]= Substituting this result into the differential rate expression for P yields: APs a(S = [P lL kT] =h[A] Now, adding the steady-state expressions for [A-] and [B-] yields: 0=2k,[A,]-26[4 JBI [As]Chapter 36/Complex Reaction Mechanisms 36.21) The overall reaction for the halogenation of a hydrocarbon (RH) using Br as the halogen is RH+Br, —>RBr+HBr. The following mechanism has been proposed for this process: Br, 9 2Br- Br-+RH—25R-+HBr R-+Br, >RBr+ Br- Br-+R-—“.5RBr Determine the rate law predicted by this mechanism. Rate = [Br-][RH] [HBr] dt Applying the steady-state approximation to the intermediate species Br and R yields: d[Br-] dt = 2k, [Br, ]-&, [Br-][RH] +4,[R -][Br,]~&, [Br-][R-] at =0=4 [Br][RH]-4[R ][Br]-4,[Br-][R] Adding the last two equations yields: 0= 2k, [Br, ]—2k,[Br-][R-] ° [8] eras) Using the steady-state approximation for dj Yt ignoring the term corresponding 10 the termination step yields: O= [Br J[RH]-4,[R [Bs] &,[Br][RH]=4 [R][Bs] oP ee =i) ( Fem ie ie O15Chapter 36/Complex Reaction Mechanisms Finally, the rate of reaction is then: 36.22) The chlo I chloride, C,H,Cl+ Cl, —+C,H,Cl,, is believed to proceed by the following mechanism: cl, 492¢1 C1-+C, HCl" 9, HCl, C,H,Cl, -+Cl, > C,H,Cl, +Cl- CHCl, -+C,Hy |, - stable species Derive the rate law expression for the chlorination of vinyl chloride based on this mechanism. The rate of vinyl chloride production is d{C,HCh} BOT 4, fener, J[ch] ‘The rate expressions of the intermediates are ACH] =k,[CI-][C,H,Cl]-&, [C,H,Cl, -][Cl, ]-2k, [CHCl y ac). 2k, [Cl,]—& [CL-][CH,CI] +4; [C,H,C1, ][C1,]=0 Adding the two preceding equations yields: 916Chapter 36/Complex Reaction Mechanisms Substitution of this expression into the differential rate expression for vinyl chloride yields: (8) ‘est feu -«(4) {a.]? 36.23) Determine the expression for fractional coverage as a function of pressure for the dissociative adsorption mechanism described in the text in which adsorption is accompanied by dissociation: Ry(g) + 2M (surface) ==? 2RM (surface) ‘The rate of absorption is second order with respect to the particle and site concentrations: 40 PN(I-0)? dt The desorption rate is also second order with respect to the number of occupied sites, thus: At equilibrium K,PN (1-0) =k, NO KP(1-0)' =6" => VKP = 6(1+ VKP) And o- YEP here kK 14VKP 36.24) The adsorption of ethy! chloride on a sample of charcoal at 0°C measured at several different pressures is as follows: Pengcr ort) Voas (mL) 20 3.0 50 38 100 43 200 47 300 48 Using the Langmuir isotherm, determine the fractional coverage at each pressure and Viy. 917Chapter 36/Complex Reaction Mechanisms The Langmuir equation can be written as Ri, And the fractional coverage, 6, is simply the ratio of adsorbed volume to the volume of maximum adsorption (V_): ae Vn Therefore, V» is required to determine the fractional coverage versus pressure. ‘The plot of £ vs. P should yield a straight line with slope equal to the inverse of V», This plot is as follows: 70 o y= 0.198204 3.07 PIV (torr/mL) P (torr) ‘The equation for the best-fit line is: Feo 198 mL"(P) +3.07 torr mL" ‘Thus, the Vm value is 1 slope =5.04 mL O18.Chapter 36/Complex Reaction Mechanisms. With Vn, @can be determined resulting in the following: P (atm) o 20. 0.595 50. 0.754 100. 0.853 200. 0.932 300. 0.952 36.25) Given the limitations of the Langmuir mode\, many other empirical adsorption isotherms have been proposed to better reproduce observed adsorption behavior. One of these empirical isotherms is the Temkin isotherm: Vj. =FlnsP, where V is the volume of gas adsorbed, P is pressure, and r and s are empirical constants. a) Given the Temkin isotherm provided, what type of plot is expected to give a straight line? b) Use your answer from part (a) to determine r and s for the data presented in Problem P36.24. a) ‘The Temkin isotherm can be manipulated to the form Vi =rin(P)+sIn(s) Therefore, a plot of Vai: versus In(P) is expected to yield a straight line. b) The data in the preceding problem plotted as Vag versus In(P) results in the following: a re a z. a , : ale e in(P) 919Chapter 36/Complex Reaction Mechanisms Best fit to the data by a straight line yields the following equation: .674 In( P) + 1.083 ‘The slope of the line is equal to r r= 0.674 mL rr, s can be determined as follows: Loro6re -99 torr | 36.26) Use the following data to determine the Langmuir adsorption parameters for nitrogen on mica: Vou. (emt g") ~[P Cor) 0.494 21x10" 0.782 4.60 x 10° 1.16 1.30 x 107 ‘The experimental values for Vj are in units of em’ or mL per gram of mica. As illustrated in the example problem in the text, the following plot is used to compare the data to the prediction of the Langmuir model: ) 1 ale P)Y, A plot of (Vaas)' versus (P)"' is as follows: 2506400 y= 0.0029 + 0.6817 2,006+00 1.006400 a 10.006+00 (0.006400 1.008402 2.006402 3.008402 4.006402 s.00E+02 P* (torr) 920Chapter 36/Complex Reaction Mechani Best fit to the data by a straight line yields the following equation: 90x10" torr g om ( ! Jeon gem P The maximum adsorption volume is, ~ 0.642 g em" 56cm’ g! ‘The K value is, (2.9%107 torr g em™)(1.56 em’ g) =221 torr P36.27) Many surface reactions require the adsorption of two or more different gases. For the case of two gases, assuming that the adsorption of a gas simply limits the number of surface sites available for adsorption, derive expressions for the fractional coverage of each gas, If we assume that the only effect of the gases is to limit the number of sites available for adsorption, then the Langmuir model yields the two following relationships: 48, = BaP (1-8-8) Ky. = kyaP, (1-8, -8,) where ky, kis and @are the rate of adsorption, desorption, and fractional coverage, respectively. In addition, the subscript denotes either gas | or 2 in the two gas mixture. The two equations can be solved to yield 0} and @, as follows: KR, "1+ KP+K,P, KP a, 1+ KP +K,P, 36.28) Sunburn is caused primarily by sunlight in what is known as the UVB band, or the wavelength range from 290 to 320 nm. The minimum dose of radiation needed to create a sunburn (erythema) is known as an MED (minimum erythema dose). The MED for a person of average resistance to burning is 50.mJ cm”. 921Chapter 36/Complex Reaction Mechanisms ) Determine the number of 290-nm photons corresponding to the MED assuming each photon is absorbed. Repeat this calculation for 320-nm photons. 6) At 20° latitude, the solar flux in the UVB band at the surface of the Earth is 1.45 mW em”. Assuming that each photon is absorbed, how long would a person with unprotected skin be able to stand in the sun before acquiring one MED? a) The energy released by 1 photons is given as he. z EA The EB n For A= 290. nm (50.0107 J) (290.x10” m) (6.62610 Js) (3.00x10° ms = 7.2910" photons em™ For A= 320. nm, (50.0107 J )(320.x10” m) (6.62610 Js )(3.00x10° ms =8.05x10" photons em™* b) Assuming an average person ~ 1 MED = 50.0mJ em, che MED is equal to the solar flux times a unit of time MED=¢-4 MEI ¢ __50.0 mJ em © 1.45 mi sem =345s P36.29) A likely mechanism for the photolysis of acetaldehyde is H,CHO + kv —+CH, -+CHO- CH, -+CH,CHO—*>CH, +CH,CO- CH,CO-—>CO+CH, - CH, -+CH,-—*>C,H, 922Chapter 36/Complex Reaction Mechanisms Derive the rate law expression for the formation of CO based on this mechanism. The rate is AC] 4, cu,c0] d Applying the steady-state approximation to [CHs] and [CHjCO] a{cu. OI -ta.yeu CHCHO]- [CH, ][CH,CHO] + & [CH,CO]-2k, (CH, } ACN) 9a [cH, ][CH,CHO]-&, [CH,CO] I Adding the last two equations yields: SoacoCH,CHO} = 24, [CH] [‘taentcucuel =[cH,] And using the steady-state expression for [CHsCO] in the rate expression for CO production results in the following: k,[CH,CO] = & (CH, [CH.CHO] “HCHO” a <1} [CH,CHO] 4 =k, (“s2) [cH,cHoyé 36.30) If r, =1 x 10°" sand k= 5 x 10% ', what is ®,? Assume that the rate constants for intersystem crossing and quenching are sufficiently small that these processes can be neglected. © , is related to kyand tyby the expression and kyis related to tyby k, +k, (assuming k; Ty, , are small ) 1, =1x10" s and k, =5x108 923Chapter 36/Complex Reaction Mechanisms ‘Thus 1 ak, +5x10" 5! 1x10" and 7, =1x10" s'-5x10" s! k, =9.5x10" 5! And finally, 5x10" s!-1x10" 95 36.31) The quantum yield for CO production in the photolysis of gaseous acetone is unity for wavelengths between 250 and 320 nm, After 20 min of irradiation at 313 nm, 18.4 em? of CO (measured at 1008 Pa and 22°C) is produced. Calculate the number of photons absorbed, and the absorbed intensity inJs' ‘The number of moles of CO is calculated using the ideal gas law: lam) 1008 P —— \(0.0184 L (100 “(ae Pa) ) (0.0821 L atm mol! K')(295 K) 50x10 mol This corresponds to 4.55 x 10'* molecules or 4.55 10'* photons that have resulted in reaction. The intensity is then determined by: o£ ne (6.626%10™ J s)(3.00%10* m s"')(4.55%10")/ ¢ mi ta nid nbn) some) 1=241x10° Is! P36.32) If 10% of the energy of a 100-W incandescent bulb is in the form of visible light having an average wavelength of 600 nm, how many quanta of light are emitted per second from the light bulb? Assuming that 10% of energy from the bulb is in the form of visible light, the power of the light is given by: P, =0.100(P) =0.100(100. W) =10.03s' 924Chapter 36/Complex Reaction Mechanisms The energy per photon at 600. nm is: 6.626x10™ Js)| 600.x10? m =331x107 J Therefore, the number of photons per second is given by: 10.03s! 3.02x10” photon s 3.1310 J photon P36.33) For phenanthrene, the measured lifeti quantum yield is 0.12, and the phosphorescence quantum yield is 0.13 in an alcohol-ether class at 77 K. ‘Assume that no quenching and no internal conversion from the singlet state occurs. Determine kp, kic!, and kis Vk, 1¢ of the triplet state (r, ) is 3.3 s, the fluorescence Assuming no quenching or internal conversion, then k © and k,t, =, +k. an k, fy thee 4 he Since ®, = 0.12, then Since , = 0.13 and r, =3.35, then 0.13 1, 3358 K, =3.88x107 925Chapter 36/Complex Reaction Mechanisms Now 88x10 0.13 Ki, = 0.260 s' 36.34) In this problem you will investigate the parameters involved in a single-molecule fluorescence experiment. Specifically, the incident photon power needed to see a single molecule with a reasonable signal-to-noise ratio will be determined. a) Rhodamine dye molecules are typically employed in such experiments because their fluorescence quantum yields are large. What is the fluorescence quantum yield for Rhodamine B (a specific rhodamine dye) where ky = | x 10° s"' and ky = 1 x 108 s"'? You can ignore intersystem crossing and quenching in deriving this answer. b) If care is taken in selecting the collection optics and detector for the experiment, a detection efficiency of 10% can be readily achieved. Furthermore, detector dark noise usually limits these experiments, and dark noise on the order of 10 counts sis typical. If we require a sigi i ratio of 10:2, then we will need to detect 100 counts s, Given the detection efficiency, a total emission rate of 1000 fluorescence photons s” is required. Using the fluorescence quantum yield and a molar extinction coefficient for Rhodamine B of ~40,000 M~! em”, what is the intensity of light needed in this experiment in terms of photons cm™ s"'? ©) The smallest diameter focused spot one can obtain in a microscope using conventional refractive optics is one-half the wavelength of incident light. Studies of Rhodamine B generally employ 532- nm light such that the focused-spot diameter is ~270 nm. Using this diameter, what incident power in watts is required for this experiment? Don’t be surprised if this value is relatively modest 108s! a) k, =1x10? s" and k, Assuming no intersystem crossing and quenching, then ky k, +k, Lx10’s 1x10? 51 1x10" s! =0.91 b) Excitation rate constant required given the signal to noise constraints and the fluorescence quantum yield is: __ 1000 photons s oe 091 1100 photons s! 926Chapter 36/Complex Reaction Mechanisms The excitation rate constant is equal to the product of light intensity and the absorption cross section, and the absorption cross section is related to the molar extinction coefficient: 2303(40,000) em? mol 1100 photons s ' = /] ~ 6.022x1 mol! 1100 photons 5" = /(1.53x10-" em’) 7.19x10" photons cms c) The focused spot size is: d) area | 2.70x10 ‘cm ) =5.72«10"em? 2 J Multiplying the required intensity by this area yields the required power in terms of photons ) P= I (area) = 7.1910" photons em? s(5.72%10-" en =4.1 x10” photon At 532 nm, the energy per photon is: he _ (6.626x10™ Js)(3.00x10* ms a 332x10” m 3.74x10"" J photon! Therefore, the power in Watts is: P=4.11%10" photons s'(3.74x10-” J photon”) =1.53x10" W= 53nW P36.35) A centeal issue in the design of aircraft is improving the lift of aircraft wings. design of more efficient wings, wind-tunnel (ests are performed in which the pressures at various parts of the wing are measured generally using ooly a few localized pressure sensors. Recently, pressure- sensitive paints have been developed to provide a more detailed view of wing pressure. In these paints, a luminescent molecule is dispersed into an oxygen-permeable paimt and the aircraft wing is painted. The wing is placed into an airfoil, and luminescence from the paint is measured. The variation in O2 pressure is measured by monitoring the luminescence intensity, with lower intensity demonstrating areas of higher O2 pressure due to quenching. 4) The use of platinum octaethylporphyrin (PtOEP) as an oxygen sensor in pressure-sensitive paints was described by Gouterman and coworkers [Review of Scientific Instruments 61 (1990), 3340]. In this work, 927Chapter 36/Complex Reaction Mechanisms a I, P the following relationship between luminescence intensity and pressure was derived: -+= 4+ 2), where Jp is the fluorescence intensity at ambient pressure Pp, and [is the fluorescence intensity at an arbitrary pressure P. Determine coefficients A and B in the preceding expression using the Stern—Volmer 5 1 . ; vet f equation: Ky =— =k) +k, [Q]. In this equation z, is the luminescence lifetime, &; is the luminescent t rate constant, and ky is the quenching rate constant, In addition, the luminescent intensity ratio is equal to the ratio of luminescence quantum yields at ambient pressure, @, , and an arbitrary pressure, ® : b) Using the following calibration data of the intensity ratio versus pressure observed for OEP, determine A and B: iil PIPy Il FIPy 1.0 1.0 0.65 0.46 0.9 0.86 0.61 0.40 0.87 0.80, 0.55 0.34 (0.83 A 0.50 0.38 077 0.65 0.46 0.20 0.70 0.53 0.35 0.10 ) Atan ambient pressure of I atm, fy = 50,000 (arbitrary units) and 40,000 at the front and back of the wing. The wind tunnel is turned on to a speed of Mach 0.36 and the measured luminescence intensity is 65,000 and 45,000 at the respective locations. What is the pressure differential between the front and back of the wing? a). Starting with the version of the Stem-Volmer Eq. provided in the problem: k, +k, [Q] ‘The luminescence quantum yield can be expressed in terms of ky and kiya a8: ‘Therefore: Kho P = +t | Kt k PK tk RR on 928Chapter 36/Complex Reaction Mechanisms b) The plot of (1p/1) versus (P/P0) is as follows: iy PIP, Best fit by a straight line to the data yields the following equati P .697 0.312 (4): 3 Thus, A= 0.312 and B= 0.697. ©) (2)_@-}e ( Pron ~ Pract (See 2) <0 nat ) 65,000 43,000 Tam (Pi ~ Prat 0.120 = 0.697] tet ( Tatm -0.172 atm = P, -P, nt — Prac P36.36) Oxygen sensing is important in biological studies of many systems. The variation in oxygen content of sapwood trees was measured by del Hierro and coworkers [J. Experimental Biology 53 (2002), 559] by monitoring the luminescence intensity of [Ru(dpp)s}” immobilized in a sol-gel that coats the end of an optical fiber impianted into the tree, As the oxygen content of the tree increases, the 929Chapter 36/Complex Reaction Mechanisms luminescence from the ruthenium complex is quenched. The quenching of [Ru(dpp)s}°" by O2 was measured by Bright and coworkers [Applied Spectroscopy 52 (1998), 750] and the following data were obtained: il % Or 3.6 12 - 48 20 78 47 122 100 a) Construct a Stern—Volmer plot using the data supplied in the table. For [Ru(dpp)s"" p= 1.77 x 10°5"', what is kg? b) Comparison of the Stem—Volmer prediction to the quenching data led the authors to suggest that some of the [Ru(dpp)s]** molecules are located in sol-gel environments that are not equally accessible to Op. What led the authors to this suggestion? ‘The Stern—Volmer plot of interest is of the form: ssion kyis the rate constant for luminescence, and k, is the rate constant for ratio versus %O> should yield a straight line: In the above expr quenching. Therefore, a plot of the inter Y= 0.0959% + 2.8078 ° 0 0 60 © 100 120 sOxygen 930Chapter 36/Complex Reaction Mechanisms Although the data are fairly well fit by a straight line, there is noticeable curvature of the data, This curvature led the authors to suggest that more than one environment may be present. 26.37) ‘The pyrene/coumarin FRET pair (ro = 39 A) is used 10 study the fluctuations in enzyme structure during the course of a reaction, Computational studies suggest that the pair will be separated by 35 A in one conformation, and 46 A in a second configuration. What is the expected difference in FRET. efficiency between these two conformational states? Using the above expression to calculate the difference in FRET efficiency: raf { 35 \— kgf { 46A by ey oe ea Dol (i) J 1386 = 0.39 26.38) In a FRET experiment designed to monitor conformational changes in T4 lysozyme, the fluorescence intensity fluctuates between 5000 and 10000 counts per second. Assuming that 7500 counts represent a FRET efficiency of 0.5, what is the change in FRET pair separation distance during the reaction? For the tetramethylrhodamine/Texas red FRET pair employed, ro = 50 A. Calculating the efficiencies corresponding to 5000 and 10000 counts/second: Eff (7500) = 0.5 Eff (5000) _ Eff(5000) _ 5000 counts s Egf(7500) «0.5. 7500 counts s* Eff(5000) = 0.333 Eff (10000) _ FfF(10000) _ 10000 counts s" Eff(7500) 0.5 7500 counts 5” Eff (10000) = 0.667 931Chapter 36/Complex Reaction Mechanisms Using these efficiencies, the corresponding separation distances can be determined: Eff (5000) = 0.333 (S0A)" 0.333 +(S0A) Eff (5000) r=S6A Eff (10000) = 0.667 7 +(S0A)" Eff (10000) ee r=4d A Ar=56-44 A=12 A 26.39) One complication when using FRET is that if fluctuations in the local environment can affect the Sy-S; energy’ gap for the donor or acceptor, Explain how this fluctuation would impact a FRET experiment, The FRET efficiency will be lower if the energy for the donor and the acceptor are further apart. Lower efficiency will result in greater uncertainty in the value ofr. P26.40) In the Marcus theory for electron transfer, the reorganization energy i and solute contribut energy is given by: partitioned into solvent ns, Modeling the solvent as a dielectric continuum, the solvent reorganization Where Ae is the amount of charge transferred, dj and d; are the ionic diameters of ionic produets, r is the separation distance of the reactants, 1” is the square of the index of refraction of the surrounding medium, and eis the dielectric constant of the medium. In addition, (42)! = 8.99 x 10° Jm C?. a. For an electron transfer in water {# = 1.33 and ¢= 80), for which the ionic diameters of both species are 6 A and the separation distance is 15 A, what is the expected solvent reorganization energy? b. Redo the above calculation for the same reaction occurring in a protein. The dielectric constant of a protein is dependent on sequence, structure, and the amount of included water; however, a dielectric constant of 4 is generally assumed consistent with a hydrophobic environment. 932Chapter 36/Complex Reaction Mechanisms a) For the transfer of one electron (de_= 1) and ¢= 80 we obtain: ag =(1.602x10" C)' x(8.99%10 JmC?) a (6x10 m) © (6x10 m) (15x10 m) } 1.33)" (80) =3.40x10" J b) With the transfer of one electron (Ae =1) and ¢= 4 we obtain: ag =(1.602x10" CY x(8.99%10? mC) —————— or (6x10 m) (6x10 m) (15x10 m) =1.20x10- J 26.41) An experiment is performed in which the rate constant for electron transfer is measured as a function of distance by attaching an electron donor an acceptor to pieces of DNA of varying length, The measured rate constant for electron transfer as a function of separation distance is as follows: kn 2I0= 10 Zor= 10 TOT ps Distance (A) 4 7 3B 32 ! a. Determine the value for f that defines the distance dependence of the electron transfer rate constant. b. It has been proposed that DNA can serve as an electron “-way” that facilitates electron transfer over long distances. Using the rate constant at 17 A presented earlier, what value of # would result in the rate of electron transfer decreasing by only a factor of 10 at a separation distance of 23 A? a) The rate constant for electron transfer is related to distance as follows: k Br Ink, =—Br+InC In the above expression Cis a constant. ‘The final expression demonstrates that a plot of Ink versus separation distance, r, should yield a straight line with a slope equal to -/: 933Chapter 36/Complex Reaction Mechanisms 20 y=-07682x+29.901 18 10 5 12.00 v.00 22.00 27.00 32.00 r(&) Best fit to a straight line results in a slope of -0.768 A" such that b = 0.768 A“. b) Aoife 038A =f 934