chapter Enzymes 1. Keeping the Sweet Taste of Corn Tho sweet isste of freshly pieked corn (1naize) is due to the high level of sugar in the kernels. Store-bought corn (several days after picking) is not as sweet because about 50% of the free sugar is converied to starch wichin one day of picking. To preserve the sweetness of fresh com, the husked ears can be imanersed in boiling water fora few minutes (*bianched”) then cooled in cold water. Com processed in this way and stored in a freezer maintains its sweetness. What is the biochemical basis for this procedure? Answer After an eat of corn has been remeved fromm the plant, the eneyme-catalyzed conver ‘son of sugaro starch continues. Inactivation of these enzymes slows dawn the conversion to {an imperceptible rate. One of the simplest techniques for inactivating enzymes i heat denatura {Uon. Freezing the corn lowers ary remaining enzyme activity to an insignificant lew 2, Intracellular Concentration of Enzymes To approninate te actual concentration oFenyines in a bacterial cel, assume tat die cell contains equal concentrations of 1,000 different enzyies int solution in the cytosol and that each protein bas a molecular weight of 100,000. Assume alse that the bacterial cell isa oylinder (diameter 1.0 an, height 2.0 ara), thas the estevel (apecific gravity 1.20) ia 205 sole ble protein by weight, and that the scluble protein consists entirely of enzymes. Calculate the average molar concentration ef each enzyme inthis hypothetical cel Answer There are thee different ways to approach this probletn, (The concentration of total protein in the cytosol is (1.2 g'nl.(0.20) = 024 x 10°F nolan = 3.4 10“ 700,000 g/mol ‘Thus, for | enzyme in 1,000, the enayme concentration is 24x 0" a = BA x 10 ” moles of each enzyme in cell (The average mol concenczaion = Mets ofesch eresme in coll Volume of bacterial eytosol = xh = (8.14)(0.60 ptn)2(2.0 rn) = 16 pr? 1.6% 107! emt = 16 % 107 = 16% 10-1 Amount (in meles) of each enzgone in cells (0.20) (0.2 ern?) (1.6 ym?) (10° oom’) CLOO,HOT EFT TOT 38x10" 16x 10 ? rl. = 28% 1077 mol Average molar concentration = 24x 107" moVls = 24 x 10 863set Chopter 6 Enzymes (ii) Volume of bacterial extosol = 7h (8.14)(0.50 jam)*2.9 mn) = 1.6 pr’ Weight of cvtosal = (pectic gravity) (volume) = (2g/mLIC6 x 10" al) = 19% 10g Avorage waht af each protein (i 1,000, 20% whet protcin) = (9% 107 g)(020/C,000) = 9.8 x 107% Average molar conventration of each protein = (average weight/(M (volume) (3X 107" iC ghool)(15 > 10 = 24 10"% moll, = 24 x 10M 6x10 ml * ml,}(1 1/1000 mL.) 8. Rate Enhancement by Urease Tie enzyine urease enhances the rate of urea hydrolysis at pH 8.0 and 20°C by a factor of 10". Ifa given quantity of urease can completely hydrelyze a given quantity of ‘urea in 5.0 min at 20 Cand pH 8.0, how long would it take for this amnourt of urea to be hydrolyzed under the sane conditicns inthe alssence of urease? Assume that both reactions take place in sterile systems so Urat bacteria cannot attack the trea. Answer “Tune to hydrolyze urea 5.0 miny(104) Gime wily daysivy = 95x 10° yr 150 million yearst 4, Protection of an Enzyme against Denaturation by Heat When enzyme solutions are heated, there is a progressive loss of catalytic activity over tine due to denaturation of dhe enzyme. A solution of Ure enayme hexokinase ineubated at 45 °C lost 50% of its activity in 12 min, but when incubated at 45 C in the presence of a very large concentration of ore ofits substrates, it lost only 3% of its actir= ly in 12 min, Suggest why thermal denaturation of hexokinase was retarded in the presence of one of its substrates. @ ) Answer One possibility is that the BS complex is more stable than the free enzyme. This n= plies that the ground slate for the BS commplex is ata lower energy level than that for the free enzyine, tus increasing the height of He energy barre” to be crossed in passing from the native 1 dhe dexatured or unfolded stat. ‘An alernatve view Is Liat an erzyine denatures in uve stages: reversible conversion of active native enzyme (N) to an inactive unfokted state (U), folowed by irreversible conversion, tw inactivated enzgmne (D) Nu Weubsirate, 8, binds only to N, saturation with $ to ferm NS would Isave Toes free N available for eoncersicn to U or I,as the N= U equilibrium is parturbed toward N. If Nbut not NSis converted to U or I, then substrate binding will cause stabilization Requirements of Active Sites in Enzymes Carboxvpertiaase, which sequentislly removes carboxylenninal amino acid residues frcan its peptide substrates, isa single pely acids. The two essential calalstic groups in the active site are fumistied by Ars epiide of 307 amino and Gue™, I dhe carboxypeptidase chain were a perfect a helix, how far apart (in A) would Arg! and Glu?" bet (Hint: see Pig. d-da.) Explain how the two amine acid residues can eatalyze a resetion occurring in the space of» few angstromsChapter € Enzymes $-65, Answer (a). Arg!" is seperated fret Gh?” by 270 — 145 = 125 amino acid (AA) residues, From Figure 44a ve see that the a helic has 3.6 AA/turn and inereases in lenath along the major axis by 5.4 Aturn. Thus, the distance between te two residucs is Ca ANosa BG AAurn (b) Three-dimensional folding of the enzame brings the two amino aeld residues inte clase proxinnty 6. Quantitative Assay for Lactate Dehydrogenase The inuscle enzame lactate dehydrogenase eat- alyzes the reaction 000" + NADH + H* — Pyrseate Lactate NADHand NAD* are du reduce and oxidized forns, respectively, of ike coenuyme NAD. Solutions of NADH, but not NAD™, absorb light at 240 nm. This property is used to determine the concentration of NADH in golation by measuring epectrophotometricaly the ammount af ght absorbed at 240 nam by the solution. Explain how these properties of NADH can be used to design a quantitative assay For lactate dohydrogonase, Answer The reaction rate can be measured by following the decrease in absorrtion at 840 nn Gas NADHis converted tn NAD*) as the reaction proceeds. The researcher noeds fo ebtsin three pieces of information to develop a good quamitative assay for actate dehydrogenase: (© _ Detertnine Ky, values (see Box 0-1) G@)_ Measure the initial rato at several known concentrations of enzyme with saturating com centrations of NADH and pyruvate (Plot the initial rates as a function off]; the plot should be Lnear, witha slope that pro- vides a measure of lactate denyarogenase concentration. 7. Efreet of Enzymes on Reactions Whict cf the following effects would be brought about by any e! _yive catalyaing the simple rection PL Ss] (@) Decreased Hg (&) Increased ley; («) Increased Ky (A) Increased AG’; (¢) Decreased AG! (0 Moro negative AG"; (g) Ineressed te P where Answer (1), (©, (@). Bnzsmes do nat change a resetion’s equllvium consiart and thus eat= lyre the reaction in both directicns, making (b) and (g) correet. Rneymes increase the rate of a reaction by lowering the activation energy, hence (e) is correct. 8. Relation betwoon Reaction Velocity and Substrate Concentration: Michaolis-Menten Equation (@) At what substrate concentration would an erayie With a gy 6f 20.0.0") and a Ky of 0.0060 m4 ‘operate at one-quarter af its maximurn race? (@) Determine the fiction of Vis that would be ebtzined at the following substate conconteations IS]: SK 2Kiny Nd LOK;$66 Chanter 6 Enmes (e) An enzyme that catalyzes the reaction X= Ys isolated tram ovo bacterial species. The erzyines have te sun? Vays DUC diferent Ay Values forthe Substrate X. Enzyme A Nasa Kin OF 20 pM, while enzyme Bs a Ky, of €.5 4. The pit below stows the kinetics of reactions earried out ‘with the same concentration of each enzyine and with [X] = 1 gat. Which curve correspon’ to which enzyine? Answer (a). Here we want to find the value of [5] when Vy ~ 0.25 Vgage The Michaelis-Menten ‘equation is tee 80 V) = Vina witen [SYK + (5) [5] = 03%, ((b) The Michaelis-Menten equation can be rearranged to VeVi = SHKy + ISD) Into the equation gives SVG, + ISD) b.25;0r = 6.93(0.0060.m) = 1.7 x 10“ an Substituting |S} ViVinae = 0.5 Kyl BKy = 0.383 Sinulany, substituing [8] = 2h gives Was = 067 And substituting [5] = LOX gives VilVinas = 09) (Ce) The upper curve corresponds to enzame B ((XIis treater than the Ky for this enzyme), and the lower curve corresponds to enzyme A. When te initial cancentration cf sub- strate is greater than Ky, the rate of the reaction is Toss sense to the depletion of substrate at early stages ofthe reaction and the rate remains approximately linear fer a Tonge time HAPPY—=sap The researchers begin to characterize the enzyme. (a) Inthe first experiment, with [Fy at 4 na, they find that the Ve is 1.5 jas! Based on this ex periment, what is the «for happyase"? (Inelude appropriate uns.)6 Enzymes 5-67 (b)_ In another experiment, with [By] at 1 nut and [HAPPY] at 30 pa, the researchers fine that 200 ns} What isthe measured, of happyase' for its substrate HAPPY? (Include appropricte ‘nits.) () Further research shows that the purified happyase® ussd in the firs two experiments was actually contamingted witha reversible inhibitor ealled ANGER. When ANGER is cavefully remewed fom the happyase® preparation, and the twoexperiments repeated, the measired Vag in CA) is itm creased to 4.8 pars! and the measured Ky in (b)is now 15 pat, Forte inhibitor ANGER, calex- Tate the values of ard a, (2) Based on the infonnation given above, what (ype of inhibitor is ANGER? Answer (@)_Use the equation hy = Vinn/MEa] Eo = 1600 no s'4 rat @)_ Use te equation Vous = kewl Be. When [Fy VilVans = 200 mak 1400 na Foarrange the Michaelis-Menten equation, substitute for Vi Vows = (8m + ISD) 3 svat. + 8 Ky = 1898 In tis experiment, the concentration ofthe substate, HAPPY, wa8 90 50 Fy = 10 GAs shownin Table 5-9, You sare 53 fron of Val’ Bet se Va nereased by actor of 3a ~ 3 Silay Ky varies sa function of afer Ge tat Fi increased bya icon af I Swen ANGER was remaved (ty Che mbit decreased the obseried fy by) ard a’ = 8 then = 2 (2) Because bth er anita ave fected, ANGER s a mixed init ani soe for Fy 10. Applying the Michaelis-Menten Equation If Another enzyme is found that calalyzes the reaction ASB Researchers find that the Ky for the substrate A is-4 wNt and the fag i820 min (a) nan experiment, [A] = 6 rs, and the initial velocity, Vy was 480 ra min What was the in the experiment? (0) In another expecimers, [B,J] = 0.5 and the measured Wy = 5 pat min What was the [A] ws inthe experiment? (€). The compound is found ( be avery strong competitive inhibitor ef the enayme, with an a of 10. In an experiment with the same [Fy as in part (a), bata different (AJ an amount of is svldedl that reeices the rate Vy to 240 aX min”! What isthe [A] inthis experiment? (@)_Rased an che kinetic parumster given above, has thie enzyme evolved to achiews catalytic rerletion? Explain your answer briefly, using the Kinet parameter) that define eats reelection. Answer (a) Because [S] is much greater than (more chan 1000-old) fq, assume that tte measured rate ofthe reaction refleets Ving Use Ue equation Vy = Boulfal, and solve for (E) Uf = Vlcae = 4800 rat im */20 min? = 24 ra (@) AC this [Ey], the calbulated Vinge = KaylEy] = 20 min! X 0.5 pat = 10 pt nin”. Recall that A, equals the substrate conceniration at which Vj = HV The measured Vy 8 ‘exactly Mal Vises 9 [A] = Kin = 4 4s-68 u. R Chaoter 6 Enzymes {e)_ Given the samme [Ey 28 in (4), Vinx = 480 rA€ min” 7, The Vy is gain exactly half Vine (V)= 240 nae nin"), so [A] = dhe apparent or measured Ky. In the presence of an Innubitor with @ = 10, the measured Ky, = 40 UA = |S} (A) No. aul, = 03NEE % 107TH) = 8.25 108 contra Tin well below the diffusion Estimation of Vinax and K, by Inspection Although graphical methods are available for accurate determination of the Vas at Kp of an enzymne-catalyzed reaction (see Box 6-1), sometines these {quantities can be quickly estimated by inspecting values of Vy at increasing [S]. Estimate te Vays and Ko of the enzgme-catalyzed reaction far which the following data were abtained. Is} Yotuntnin) {si 00 Ye uulind 25x10 28 ato rrrs 40x10 40 recto 128 1x10" n 2x10 139 2x10* % 1x10 140 Answer Notice bow ltile the velocity changes as the substrate concentration increases by fve- fold from? to 10 mat Thus, we can estimate a Fa. Of 140 pAUMIN. Ky, is defied as the substrate concentration that produces a velocity of ;Vaax- 07 70 animin.Inspecticn of the table indicates thax this Vy occurs at [S] = 1X 10° aC tks Ry = 1 X18 Properties of an Enzyme of Prostaglandin Synthesis rostaglandins are a clas of eicasanorts, fay acid dericauves with a vanety of extremely potent actions on vertebrate issues. They are re sponsible for producing fever and inflammation and its associated pain, Prastaglancins are derived fron the 20-carbon fauiy aeld arachidonic ac in a reaction catalyzed by Ue enwytne prostaglandin en doperoxide syntlase. This enzyme, a cyclooxygenase, uses exyyen Le convert arachidenie acid W PGG., de immediate precursor of many different prostaglandins (prostaglandin synthesis Is Described in Chapter 21) (a). The kinetic data given below are forthe reaction catalyzed by prostaglandin endoperoxide syn- ‘hase. Fecusing here on the rst two columns, determine the Vina: afd Kim OF the enzyme Rate of formation of PCG: TAnctidosicacia)_ | Rate of formation of PGS, | with 10 mg/nkibuprtert ‘ona a) on) a5 235) 1687 1.0 322 25.25 15 363) 3049 25 na 3704 35 aap 3891 () Ibuprofen is an inhibiter of prostaglandin endoperoxide synthase. By inhibiting the eynthesis of prestaglanding, ibuprofen reduces infaramstica and pein, Using the date in the frst ard third columns of the table, determine the type of inhibition that ibuprofen exerts on prosiaglandin ondopenuxide synthaseChapter 6 Enzymes $69 Answer {a) Calculate the reciprccal values for the data, asin parentheses below, and prepare a double-reciprocal plot to determine the kinetic paramevers. No wth 10 mgmt ibuprofen 4 (ei) {oie {S1mu) AS) (owt) | CaN nit) (0 tei) 03 2.0 23.5 (0083), 15.67 (0.0500) 1044.0) 2.20031) 23.25 0.0396) 130067 3.90027) 39.49 (0.0328) 2510.40) 41.8 (002) 37.04 00.0270), 351028) 44.0 (0023) 38.91 (0.0257) Sony mehr 6 ‘The intercept on the vertical axis = —1/Vgg aie Ute intercept on the horizontal axis = — UK Prom these values, we can calculate Fnac and Ky Wa = ~ 0.0104, 0H Pgs = BB nt/inin UK = 1.7, and Kg = 0.59 108 (b)_buprafen acts as a compesttive inhibitor. The double-reciproeal plot (wih InhibiIor) shows that, in he presence of ibuprofen, (he Ving Of Ue reaction is unchanged (Ae ie {ervept on Ue 1/% axis isthe sane) and Ky is Increased (~1/Ry is closer 10 Ue origi). 15. Graphical Analysis of Vinac td By The fuilowing experimental data were eollecved drlag a suey of the catalytic activity of an intestinal peptidase with the substrate glycylycine: Glycyllycine + HO —* 2 glycine Proguet formed Product formed sium ‘navi {51 (om ‘java. 15 021 40 038 20 024 80 0.40 50 028 160 0.458.70 Chapter 6 Emymes Use graphical analysis (see Box 6-1) vo detenmlne the Ky, and Vax for this enzyine preparation and substrate. Answer As describe! in Box 6-1, the standard method is to use Vy versus [5] data to calcu: Tate 1% aed 143} Vo(umovinin) | 11M (minfymon) | 181 rm) Ms) eu") oat a8 15 067 2a 42 20 050 028 36 30 033 oa a0 410 025 oo 25 80 013 ous, 22 160 006 Graphing these values gives & Lineweaver-Burk plot. From the best straight line through the data, the intercept on the horizontal axis = —1iy and the intercept on the vertieal axis = 1Wpyx- Prom these values, we can calculate Kp and Vay Ky, = “045, ad Ky, = 2.2 100 LW = 72.0) aNd Vinge = 0.60 pmeliain 14. The FadieTTofsiee Equation One (arsforiation of the Mihelis-Mengen equation Is he Lineweaver-Burk, or double-reciprocal, equation. Multiplying both sides uf the LineveaverBurk equation by Vaux and rearranging gives the adie-Hottec equation: n= (Kel gy + Fo A plocor Vy vs. VylS) for an exzyine-cavalyzee reaction is shown below. The curve labeled "Siope = Ay" was obtained in de alsence of inhibitor Which of the ether curves (A, B, er 6) shows the enayme aetiity when a competitive inhibitor is added to the reaction mixture? Hint: See Equation 6-20Chapter 6 Enaymes—S.71 Answer Curve A shows competitive inhibition. Vay, for Ais the same as for the normal ‘curve, as seen by the identical intercepts on the V axis. And, for every value of [S} (until maximal velocity is reached at saturating substrate levels), Vis lawer for curve A than for the normal curve, indicating corapettive inhibition. Note that as [S] increases, VIS) decreases, so hat Vpge—UHet is, the Vy ak the highest (saturating) (S}—is found at the intersection of he ‘curve at the y ax’. Curve G, while also having an identical Vg, shows higher Vy values for ‘every [S| Cand for every VefS)) than the normal curve, which is rot indicative of inition. ‘The lower Fug for curve B rules aut competitive labo. 16. The Tumover Number of Carbonic Ankydrase Carbonic anhydrase of crythrvoyies (M, 99,000) has eno ofthe highest tumover numbore we know of It catalyzes the reversible hydration ef CO H.0 + C02 = H,00, ‘Taio io an important process in the transport of CO, from the tisques to the lungs. IF 10.0 pg of pure ‘carbonic anhydrase catalyze the Hydration of 0.30 g of COs in 1 min 3¢ 37 °C at Uys wha isthe tumover number (ka) of earbonie anbysrese (in units of min”)? Answer The turmoser number of an enzsime isthe number of suastrate molecules trans formed per unit time by a single enzyme molecule (ora single catalste site) when the enzyme is saturated with substrate ky at = Vina where E, = total males of active site. We can convert the values given in the problem into & turnaver nuraber (min converting the weights of enzyme and substrate to molar amounts: 030 w/in Hi wiwol ») by “ene (toles of COs/min) = 6.8 10-* medi (20.0 pa) (1 9/10 pa) ‘30,000 gnol B10" ol Amount of enzyme (moles) = ‘The tumover number is obtained by dividing moles of COyinin by roles of enzyme: fg = SS mod 9 1 in! 33% 16, Deriving a Rate Equation for Competitive Inhibition The rate equation for an. enzyme subject to cunpetiive intbiton is Ye ISL Oe + (ST Begining with a new definition of total enzyine as (E41 = (8) + (ES) + (EN) ‘and the definitions of « and Kj provided in che text, derive the rate equation above. Use the derivation fof the Michaclis Menten equation 2s a guide. Answor Tho basic esourmptions used to derive the Michaelis-Menten equation sill held. Tho reaction is at steady state, and the overall rate is determined by ka\BS) @ With the ccenpetitive inhibitor, now to be added, the goal again is to describe Vin tormns of the measurable quantities (Fy, [8], and [I]. In the presence of mibitor,S72 Chapler 5 Eneymes (FJ = FS} + [B] + (21) (b) We rst suve for EAs we have seen tra a KT oem ‘Substituting for [EI] in (b) gives ted = (es) + ey + 2 © and simplifying gives fe) = es) + tei. +2)= (Bs) + (le a) where « descrites the effect of the competitive inhibitor. [2] inthe absence af inhibitor ean be ‘obiaine from a rearrangemert of Equation 5-19 (remembering that [&4] = [ES] + [E), to give ESI py = ES © = ER © Substituting (c) into () gives - (5 ten = 88) + (I) © and rearranging and solving for [BS] sives (allS] +15) Next, substituting (g) into (a), and defining fF) = (FSI o Vosns We Bet the final equation for reac tion Velocicy in the presence of @ competitive inhibitor: VnaalS) we On TS) 17, Irreversible Inhibition of an Enzyme such as Hy, Cu" lany enzsmes are inhibited ireversibly by heavy motel ions or Ag, whieh can react with essential sulfaydryl groups to form meresptides: Eru—SH + Ag’ —>Brz—S—Ag + HY ‘The affinity of Ag* for sulfhydryl groups isso great that Ag* can be used to titrate SH groupe quantitatively. To 10.0 ml. of a solution containing 1.0 mg/mL of a pure enzyme,an investigator added just enough AgNO, to completely inactivate the enzyme. A cota of 0.312 mol of AgNO was required. Calculate the minimum molecular weight ofthe enzyme. Why does te value obtained in this way give only the ménimum molecular weight? Answer An oquivsloncy exiets between millimeles of AgNO, required for inactivation and millimoles of —SH group and thus, assuming ene —SE group per erzyme molecule, milimoles of enzyme CO mpm 160 mb) [ininimum H,)Gnig/monol) (1.0 mgmt.) (10.0 mb) 0842 10 mend ‘This is che mirtinmem molecular weight because it assumes only one titratable —SH greup per enzyme molecule 0342 % 107% mmol = ‘Thus, tue atone My = = 29x 10" = 29.00018. Hi’. Chapter Eraymes $73 Clinical Application of Differential Enzyme Inkibition Human blood serum cortains a class of cenaymes known as acid phosphatases, which hydrolyze biological phosphate esters under slightly acidie conditions (pH 5.0) R-O—PO}" + HO —> ROH + HO—POF! Acid phosthatases are produced by erythrocites, the liver, Kine, spleen, and prostate gland. The er- zzome of the prosiate gland is clinically important because its increased activity in the blood can bean indication of prostate cancee. The phosphatase from the prostate gland is strongly inhibited by tartrate jon, but acid phosphatases from other tissues are not. How can this information be used te develop a specific procedure for measuring the activity ofthe acid phosphatase of the prostate gland in human blood seruin? Answer Fist, measure the ‘otal acid phosphatase activity ina blocd sample in units of sano! of phosphate ester hydroiyaed per mn of serum, Next, remeasare this activity in the presence of tartrate ion at a concentration sufficient to completely inhibit the erzyme from the prostate lend. The difference between te two activities represents the activity of acid phosphatase fram the prostate gland Inhibition of Carbonic Anhydrase by Acetazolamide Carboric anhydrase is strongly inhibited by the drug acetazaamide, whieh i used asa diuretic (Le, to inerease the pruduetion of urine) and to lower excessively high pressure in the eye (due fo accumulation of irtraocular Mud) in glaucoma, Car- ‘onic anhydrase plays an important role in these and ceher secretory processes because & participates in regulating the pH and bicarbonate content of several body Mids. The experimental curve of initial reac tion velecity (as percentage of Vp) versus [S] for the carbonic anhydrase reaction is Mustrated below (upper curve). When the experiment is repeated in the presen: of acetzzolamide, the lower curve is ob {ained, From an inspection of the curves and your knowledge ofthe kinetic properties of competitive and ‘mixed erayme inhibitors, determine the nature of the inhibition by acetazolamide. Exphin your reasening, Noinhibitor rr Slim Answer Tie graph gives us several pieces of information, Fis, the itor prevents te enryme tram achieving the same Fue 28 i Ce abeence of inhibior Seccnd. the overall shape Of de two curves is vty similar: at ay [She ratio of the wu velocities Csintbbon ithe Saine, Thin, the veWety does ne ekarge very mich stove [5] = Lin, 30 aL MUCH Nghe [5] Ue observed velociy Is esertally Vg foreach curve. Fourth it we estima the (at whch ‘Ws sachets vale neaty Henao bth curves. Neneanpedtive ition, a Special urn of nixed ition thats rarely observe, alters the Vaya of enzymes bit aves ZB, unclanged. Ths, acetazolamide acs as a noncompetitive (nixed inhibitor of carbonic aniydraeS74 Chapter © Enzymes 20. The Effects of Reversible Inhibitors Devive Ue expression for the effect of areversitle mbubitor ‘on ouserved Fy (appArENL Kyy = afi’), Stark wi Equation 6-30 and the statement Uhat Aappazent Ky is equivalent. (w the [S}ab WW Vy = Voas/2at Answer Equation 0-20 is Vas * ky BT Or Vy = Vas X [SCOR + ee1S). Thus; the [S} at which Vi = Vogu/2xis obtained when all the terms fon the right side of the equation except Vays equal 4a’ SVCan, + a(S) = Ja’ Wo can now sve this equation for [S: 2a'lS| = ay + a''S} 2el(S} ~ a(S] = ak eS] = ak, 18) = atigla” Thus, obsteved By = aKy/e! 21, pH Optimum of Lysozyme Tre acsve sie of lysuzyine Contains two amine acid residues essential for ‘caialysis: Glu" and Asp". The pF, values ofthe carboxyl skde chains of these residues are 59 and 4.5, respectively. What isthe lonizatien state (protonated ar deoreconated) of each residue at pH5.2, Whe Dl optimum of lysozyme? How can dhe ionization states of these residues explain the pll-activty pro- Iie of lysezyrne shown below? oH Answer At al inidway between the two pk, values (pH 5.2), the side-chain carboxy! group ‘of Asp", with the lower pk, (4.5), is mainly depratonated (—COO), whereas Gla”, with the higher pi, (59; the stronger base), i proiorated (—COOH). At pH values below 52, Asp’ Devomes protonated and the aetvity decreases. Similarly, at pH values above 52, Gh be- ‘comes deprotonated and the activity aso decreases, The pi-activity profile suggests that ‘maximum catalyule acuvity occurs a a pH midway between the pA, values of the two acide ‘2roups, when Giu”is protonated and Asp’ s deprotonated. 22. Working with Kineties Go lo the Living Graphs for Chapier 6 (@) Using the Living Graph for Equation 6-9, ereate a V versus [S] plot. Use Vgge = 100 ya's, and Ky = 10 pi. How much does Vy increase when [8] is doubled, from 0.2 to Ul pat? What is Vy when [] = 10 pit? How much does the V4 increase when [S] tcreases from 100 to 200 Xe? Observe haw the graph changes when the values FoF Vx OF Byy A€e halved oF doubled,Chapter 6 Enaymes (b) Using the Living Graph for Equation 630 and the kinetic parameters in (a), create a pt in ‘which ott a and a are I.0. Now observe how the plot changes when a = 2; when a’ and when a = 20 and a’ = 3. Ce). Using the Living Graphs for Equation 6-80 and the Lineweaver-Burk equation in Box 6-1, create Lineweaver-Burk (double-reciprocal) plots forall the cases in (a) and (1b). When @ = 2.0, does Ue iniereept move Lo the right or lo Ue TeR? Ha = 20 and a” = 3.0, does the x inversept move tothe right or to te fet? 8.0; Answer (a). When [5] increases from 02 to 0.4 wh, Vy increases by a factor of 1.96. When [S] = 10 1, V) = 50 jac 2°, When [S]inerenses from 100 10 200 pa, V, ineroases by & actor of 14s, () When a = 2.0, the ear ie shifted to tho right as the Ky is increased by a factor of 2 When a’ = 3.6, the asymptote of the curve (the Ving) declines by a factor of 3. When ef = 20 and a" =, the curve briefly rises above the curve where both « and a! = 1.0, due to adesline in Fy. Hewwever, the asymptote is lower because Vu declines By a factor of 3. CO) When a= 2.0, the 2x intervet moves to the right. When o Intercept moves to the lef. 20 and a! = 80, the a Data Analysis Problem 23. Exploring and Engineering Lactate Dehydrogenase Fxsiining the structure of an enzyme re sults in hypotheses about the relationship between different amino acids in the protein's structure and the proteir’ function. One way to test these hypotheses is to use reccanbinant DNA technclogy to generate mutant versions of te enzyme and then examine the structure and funetion of these altered orms. The technology used to do this is described in Chapter 9. One example of Us kind of analysis isthe work of Clarke and colleagues on the enzyine lactate denydrogenase, publisted in 1989. Lactate dehydrogenase (LDH) catalyzes the reduction of pyruvate with NADH to forin lactate (see Section 14.3). A schematic of the enzyine’s acuive site Is shown below; he pyruvate & in he center Icntadasdrogenane ‘que é AN ant of Sat a ae ! H.c—¢-08 any H ye OFF pyruvate «wae swe Lee pone Wd | oA a ony 2 NAH HaC—b- En, wf hye nee an 375576 Chapter 6 Enzymes ‘The reaction mechanism is similar to many NADH reductions (Pig. 19-24); i is approximately the revere of stepa 2 and 2 of Figure 14-7. The transition state involves strongly polarived carbeny! sroup of the Fyruvaie molecule as shown belew Hy (4) A.mutane form of LDH in which Arg" ing and 0.07% of the aetivicy of wikl-lype enzyme, Provide a plausible explanation for he ef of his mutation (€H) A euucane Form FL.DH in which Ang!" Is replaced widh Lys shows only 0.05% oF dhe wild-type evel of substrate binding, Why is this dramatic effect surprising? Ce). Inthe crystal strucuare of LDH, the guanicinnurn group of Arg" and the carboxyl group of pyru= vale are aligned as shown ina co-planar “Torked” configuration, Based on this, provide a plausible explanation forthe dramatic etfeet of subsutueing Arg” wath Lys, 4) A mutant form of LDH in which Ile is replaced with Gin shows reduced binding of NADH. Pro- Vide a plausible explanation for this result 1s replaced with Gin shows only 5% of the pyruvate bind- eas Clarke and calleagues also set out to engineer a mutant version of LDH that would bind and ro- duce oxsloseetate rather than pyravste. They made a single substitution, replacing Gla!" with Arg the resuiting engyime would reduce oxaleacetate to malate and would no longer rechce pyruvate to lactate, They had therefore concerted LDH to malate dehvaragenase, Ce). Skoten whe active site oF this murant LDH wih oxaloacetate bound. (8) Provide a plausible explaration for why this mutant enzyine now "prefers" oxaloacetate Instead of pyruvate, (a). The authors were surprised that substituting « larger amino act in the active sit allowed a larger substrate to bind. Provide a plausible explanation for this resul. Answer (a) tn dhe wid-ype enzyme, the substrate held in place by a hydrogen bond an anion: dipole inveracton between the charge side chain of Arg! and the pole carbonyl of pyruvate. During catalysis, the charged Arg side chain also stabslizes the polarzed tabonyl transition site, In Ute mutant, the binding is ceduced to just a hydrcgen bond, sulstrate binding is weaker, and ionie sabiization ofthe transition state iso, reducing calaltic activi. (b)_ Because Lys and Ang are roughly the sanne size and havea sila pesitive charge, they probably have very simlar properties. Furthermere, because pyruvate binds to Arg!" by {resumnably) anionic interaction, an Arg to Ly nucation would probably have Tile effect on substrate Lining (6) The “forked” aerangement aligns two positively eharged groups of Arg residues with the negatively charge exygens of pyruvate and facilcates wo combined hydregea-bond and ion-dipol interactions. When 13s is present, only ore such combined hydrogen-bond and le-diple interecion is possible, thus reducingthe strenath ofthe Interaction. The positioning of te substrate is less precise €@) Te" interacts hydrophobically withthe ring of NADEL This (pe of interacsion isnot possible with the hydrophilic side chain of GnDemsee 2/68 nam oie co oma gsi maton kon References «© o © ChoptorS Eneymes 8.77 The structure is shown below. ‘The mutant enzyme rejects pyruvate because pyravate’s hydrophobic methyl group will ‘net interact with the highly hydrophilic guanidinium group of Arg!"®. The mutant binds coxaleacetate because ofthe strong ioric interaction between the Anz!” side chain and the carboxsl of oxaloacetate. ‘The protein must be flexible enough to arcemmodate the addled luk cf the side chain an te larger substrate. Clarke, AR, Atkinaan, Te Holle, (1980) vom anal sptesis new land bing ses an he ce dey drogenaeanevr, Pat 1-Pets Bice, So. 44, 10-106. 0 sets ew ga ining eso te