Enzymes 1. Keeping the Sweet Taste of Corn ‘The sweet taste of freshly picked com (maize) 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 converted into starch within one day of picking. ‘To preserve the sweet- ness of fresh corn, the husked ears can be immersed in boiling water for a few minutes (“blanched”) ‘then cooled in cold water. Corn processed in this way and stored in a freezer maintains its sweetness. What is the biochemical basis for this procedure? Answer After an ear of com has been removed from the plant, the enzyme-catalyzed conver- sion of sugar to starch continues. Inactivation of these enzymes slows down the conversion to an imperceptible rate. One of the simplest techniques for inactivating enzymes is heat denatu- ration, Freezing the corn lowers any remaining enzyme activity to an insignificant level. 2. Intracellular Concentration of Enzymes ‘To approximate the actual concentration of enzymes in a bacterial cell, assume that the cell contains equal concentrations of 1,000 different enzymes in solution in the cytosol and that each protein has a molecular weight of 100,000. Assume also that the bacterial coll is a cylinder (diameter 1 jam, height 2.0 jum), that the cytosol (specific gravity 1.20) is 20% solu- ble protein by weight, and that the soluble protein consists entirely of enzymes. Calculate the average molar concentration of each enzyme in this hypothetical cell, Answer There are three different ways to approach this problem. @__ The concentration of total protein in the cel is (2.2 gmb(02) a _ a “100,000 ghmot 7 024 X 10"S molinLs = 2.4 x 10°? ‘Thus, for one enzyme in 1,000 24x 107m the enzyme concentration = “7 = 24 10° mol, = 2m (i) The average molar concentration = arth 57X10 L Amount (in moles) of each enzyme in cell is (0.20)(1.2 glem?)(1.57 wmm®) (107mm? (200,000 g/mol) (100,000) 3.77 x 10°? mol “Tat x 10 = 24x 107° moVL ~ 2 pm Volume of bacterial cytosol = 8.77 x 10° mol Average molar coneentrationChapter 8 Enzymes $-49 | (ii) Volume of bacterial cytosol = rh = @.14)(0.5 pm)? wm) = 1.57 am’ = Weight of cytosol = (specific gravity) (volume) = (12 gimL)(L.67 x 10-!? mL) = 1.88 x 107" g ‘Average weight of each protein (1 in 1000, 20% wt/wt protein) = (1.88 x 107 g)(0.2)/(1000) = 3.77 x 107" g ‘Average molar concentration of each protein (average weight)/(M,) (volume) (8.77 x 10" g/(10" g/mol)(1.67 x 10-"? mL)(1 1/1000 mL) 24 X 10° mol/L = 2 pat 87 X 107 mL 3. Rate Enhancement by Urease The enzyme 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 hydrolyze a given quantity of urea in 5 min at 20 °C and pH 8.0, how long would it take for this amount of urea to be hydrolyzed ‘under the same conditions in the absence of urease? Assume that both reactions take place in sterile systems so that bacteria cannot attack the urea. Answer ‘Time to hydrolyze urea . (@minycuo" (60 mirvh) (24 h/day) (B65 days/yr) = 95 x 10° yr = 950 million years! Protection of an Enzyme against Denaturation by Heat When enzyme solutions are heated, there is a progressive loss of catalytic activity over time due to denaturation of the enzyme. A solution of the enzyme hexokinase incubated 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 one of its substrates, it lost only 3% of its activ- ity 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 ES complex is more stable than the free enzyme. This im- plies that the ground state for the ES complex is at a lower energy level than that for the free enzyme, thus éncreasing the height of the energy barrier to be crossed in passing from the native to the denatured or unfolded state. ‘An alternative view is that enzymes undergo unfolding in two stages involving reversible conversion of native, active enzyme (N) to an unfolded, inactive state (U) followed by conver- sion to the irreversibly inactivated enzyme ({): USI If substrate binds only to N, saturation with S to form NS complexes means that less free Nis available for conversion to U or I, as the N= U equilibrium is perturbed toward N. If N (free enzyme) but not NS complexes are converted to U or I, this will cause stabilization. Requirements of Active Sites in Enzymes Carboxypeptidase, which sequentially removes car- boxyl-terminal amino acid residues from its peptide substrates, is a single polypeptide of 307 amino acids. The two essential catalytic groups in the active site are furnished by Arg'*® and Glu”. (a) If the carboxypeptidase chain were a perfect « helix, how far apart (in Angstroms) would Arg'“® and Glu”? be? (Hint: See Fig. 6~4b.)i il i i | i $50 Chapter Enzymes (b) Explain how two amino space of a few Angstroms, is, separated by this distance, can catalyze a reaction occurring in the Answer (a) Arg" is separated from Glu?” by (270 ~ 145) = 125 amino acid (AA) residues, From Figure 6—4b we see that the a helix has 3.6 AA/turn and increases in length along the major axis by 5.4 A/turn. Thus, the distance between the two residues is (025 AA)(6.4 Aun) 36 AAAumn| 188A (b) Three- 2 glycineChapter 8 Enzymes $-53. Product formed Product formed {s1 (mw) (umovmin) {S1(mm) (umovmin) Use graphical analysis (see Box 8-1) to determine the Ky and Vinx for this enzyme prepara- tion and substrate. Answer As described in Box 8-1, the standard method is to use Vp versus [S] data to caleu- late 1/V%9 and 1/1) Yo (m/min) 1 (rminimg) Is (mu) NS} (mu) Graphing these values gives a Lineweaver-Burk plot. From the best straight line through the data, the intercept on the horizontal axis = —1/K, and the intercept on the vertical axis = UVa From these values, we can calculate Ky and Vinx Ky = 2.2 mo Vinge = 0.61 pmol/min 1, The Eadie-Hofstee Equation One transformation of the Michaelis-Menten equation is the Lineweaver-Burk, or double-reciprocal, equation. Multiplying both sides of the Lineweaver-Burk equa- tion by Vinax and rearranging gives the Eadie-Hofstee equation: Yo 2 + Vane is} A plot of Yo vs. Ve/IS] for an enzyme-catalyzed reaction is shown on the next page. The curve with a slope of ~Kq (the “normal” curve) was obtained in the absence of inhibitor. Which of the other curves (A,B, or ©) shows the enzyme activity when a competitive inhibitor is added to the reaction mixture? Ki!$54 Chapter 8 Enzymes Xa isi Answer Curve A shows competitive inhibition, Vig for A is the same as for the normal curve, {as seen by the identical intercepts on the Vj axis. And, for every value of (S} (until maximal velocity is reached at saturating substrate levels), Vo is lower for curve A than for the normal curve, indicating competitive inhibition, Note that as [S] increases, Vo/{S] decreases, so that Vyax-that is, the Vo at the highest (saturating) (S]—is found at the intersection of the curve at the y-axis. Curve C, while also having an identical Vax, shows higher Vo values for every {S] (and for every Vo/[S)) than for the normal reaction, which is not indicative of inhibition, 12. The Turnover Number of Carbonic Anhydrase Carbonic anhydrase of erythrocytes (M, 80,000) has one of the highest turnover numbers among known enzymes. It catalyzes the reversible hydration of CO»: HO + CO; == H,CO5 ‘This is an important process in the transport of CO. from the tissues to the lungs. If 10 pg of pure car- onic anhydrase catalyzes the hydration of 0.30 g of CO, in 1 min at 37 °C at Vinax what is the ‘turnover number (Ks) of carbonic anhydrase (in units of min")? Answer ‘The turnover number of an enzyme is the number of substrate molecules trans formed per unit time by a single enzyme molecule (or a single catalytic site) when the enzyme is saturated with substrate: eat = Vala where By = total moles of active sites ‘We can convert the values given in the problem into a turnover number (min™*) by con- verting the weights of enzyme and substrate to molar amount: 0.3 g/min ' O38 g/min _ 6g x 19-8 ‘gino ~ °° * 10 0 aC gO" ws) _ 30,000 g/mol. ‘The turnover number is obtained by dividing moles of COyfnin by moles of enzyme 6.8 x 10-8 mol/min “33x 1 mol Vax (rnoles of CO,/min) = ‘molinin Amount of enzyme (moles) 83 x 107" mol = 2.0 x 107 min“Chapter 8 Enzymes S55 13. Deriving a Rate Equation for Competitive Inhibition The rate equation for an enzyme subject to competitive inhibition is _ Yea) Vo" Rn + 1S) Beginning witha new definition of total enzyme as (B= 0) + (En +S) and the definitions of a and K; on p. 266, derive the rate equation above. Use the derivation of the Michaelis-Menten equation in the text as a guide, Answer The basic assumptions used to derive the Michaelis-Menten equation still hold. The reaction is at steady state, and the overall rate is determined by: Vo = ke [ES] @ With the competitive inhibitor, I, now to be added, the goal again is to describe Vo in terms of the measurable quantities (Ey), [S], and [I]. In the presence of inhibitor, (6) = (ES) + [B) + EN] ) We first solve for (EI ‘As we have seen, CB) substituting into (b), we get: (et) I=(ES|+IE)+ > ©) £0 K Ki ey ‘Simplifying gives: - (14). 7 (Bi) = (85) + (E] (1+) = [ES] + @ ‘The term a describes the effect of the competitive inhibitor. The term [E] in the absence of inhibitor can be obtained from a rearrangement of Eqn. 8-19 (remembering that [Ey] = ES] + (ED), giving: (BS) Kw Bes ©) Substituting (¢) into (@) gives _ SI K, ted= tes) + (“T © Rearranging and solving for [ES] gives: tes) - Ess _ © Kus +S Substituting (g) into (a), and defining ks{F,] = Vinx, gives the final equation for velocity in the presence of a competitive inhibitor: Vax 8] Kye + (8) w Vo$-56 Chapter 8 Enzymes 14, Irreversible Inhibition of an Enzyme Many enzymes are inhibited irreversibly by heavy-metal ions such as Hg?*, Cu?*, or Ag*, which can react with essential sulfhydryl groups to form mercaptides: Enz—SH + Ag’ —> Ena—S—Ag + HY ‘The affinity of Ag* for sulfhydryl groups is so great that: Ag can be used to titrate —SH groups quan- titatively. To 10 mL ofa solution containing 1.0 mg/mL, of a pure enzyme, an investigator added just ‘enough AgNOs to completely inactivate the enzyme, A total of 0.342 jumol of AgNO» was required. Cal- culate the minimum molecular weight of the enzyme. Why does the value obtained in this way give only the minimum molecular weight? Answer An equivalency exists: __ G0 mg/mi)(10 mL) (Ginimum Mf.) Cng/amol) _ G0 mg/n)(10 mL) 0.342 x 10° mmol ‘This calculation assumes that the enzyme cont 0.342 x 10* mmol ‘Thus the minimum M, = 2.9 x 10* = 29,000 is only one titratable —SH group per molecule. 15. Clinical Application of Differential Enzyme Inhibition Human blood serum contains a class of enzymes known as acid phosphatases, which hydrolyze biological phosphate esters under slightly acidic conditions (pH 5.0): R-O—POF- + 1,0 —> R-OH + HO—POF Acid phosphatases are produced by erythrocytes, the liver, kidney, spleen, and prostate gland. The enzyme from the prostate gland is clinically important because its increased activity in the blood is fre- quently an indication of prostate cancer. The phosphatase from the prostate gland is strongly inhibited by the tartrate ion, but acid phosphatases from other tissues are not. How can this information be used to develop a specific procedure for measuring the activity of the acid phosphatase of the prostate sland in human blood serum? Answer First measure the fotal acid phosphatase activity in a blood sample in units of ol of phosphate ester hydrolyzed per ml. of serum. Then remeasure this activity in the presence of tartrate ion at a concentration sufficient to completely inhibit the enzyme from the prostate gland. The difference between the two activities represents the activity of acid phosphatase from the prostate gland. 16. Inhibition of Carbonic Anhydrase by Acetazolamide Carbonic anhydrase is strongly inhibited by the drug acetazolamide, which is used as a diuretic (to inerease the production of urine) and to treat glaucoma (to reduce excessively high pressure in the eye due to accumulation of intraocular fluid). Carbonic anhydrase plays an important role in these and other secretory processes because it partici- pates in regulating the pH and bicarbonate content of a number of body fluids, The experimental curve of initial reaction velocity (as percentage of Vinax) versus (S] for the carbonic anhydrase reaction is illustrated on the next page (upper curve). When the experiment is repeated in the presence of acetazolamide, the lower curve is obtained. From an inspection of the curves and your knowledge of the kinetic properties of competitive and mixed enzyme inhibitors, determine the nature of the inhibi- tion by acetazol-amide. Explain.Chapter 8 Enzymes $-57 100 ‘No inhibitor V5 of Vins) g 02 0406S (sim) Answer The graph gives us several pieces of information, First, the inhibitor prevents the enzyme from achieving the same Vigx as in the absence of inhibitor. Second, the overall shape of the two curves is very similar: at any [S] the ratio of the two velocities (inhibitor) is the same. Third, the velocity does not change very much above [S] = 1 mus, so at much higher [S] the observed velocity is essentially Vax for each curve. Fourth, if we estimate the [S] at which. £2V max is achieved, this value is nearly identical for both curves. Noncompetitive inhibition, a special form of mixed inhibition that is rarely observed, alters the Vina of enzymes but leaves Ky, unchanged. Thus, acetazolamide acts as a noncompetitive (mixed) inhibitor of carbonic anhydrase. 7. pH Optimum of Lysozyme The active site of lysozyme contains two amino acid residues essential for catalysis: Glu® and Asp™. The pi, values of the carboxyl side chains of these two residues are 5.9 and 4.5, respectively. What is the ionization state (protonated or deprotonated) of each residue at pH 5.2, the pH optimum of lysozyme? How can the ionization states of these residues explain the pHT-activity profile of lysozyme shown below? 100 Activity (% of maximal) Answer At a pH midway between the two pk, values (pH 6.2), the side-chain carboxyl group of Asp®, with the lower pK, (4.5), is mainly deprotonated (—COO”), whereas Glu”, with the higher pK, (59; the stronger base) is protonated (—COOH). At pH values below 5.2, Asp becomes protonated and the activity decreases. Similarly, at pH values above 6.2, Gli®® be- comes deprotonated and the activity also decreases. The pH-actvity profile suggests that maximum catalytic activity occurs at a pH midway between the pK, values of the two acidic j groups, when Glu® is protonated and Asp™ is deprotonated,