The Journal of , Vol. 58, No. 1, 1965

Effects of on the Activity and Structure

of Dehydrogenase


(From the Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Bunkyo-ku, Tokyo)

(Received for publication, March 29, 1965)

There have been many reports (1-8) on (9) and K lot z (10). However, to interprete the effects of urea on the molecular structure the mechanism of inhibition by urea on and activity of various . Most reactions, further investigations are necessary enzymes (1-4) are known to be inhibited by since few have covered both kinetics of the en low concentrations of urea which do not zymatic reaction and physicochemical analysis denature their . The mode of inhibi of the structural changes of the enzyme protein. tion of urea on various enzyme reactions has In this paper, the effects of urea upon yeast been studied kinetically by R a j a g o p a l a n, alcohol dehydrogenase [EC 1. 1. 1. 1, Alcohol: Fridovich and Handler (3) and also NAD oxidoreductase (yeast)] have been studi by Chase's group (6-8) using rather low ed, focusing attention on the relationship be concentrations of urea. The inhibition by tween the structure of the enzyme molecule urea of various enzymes, such as xanthine and its catalytic activity. oxidase [EC], muscle lactate dehydro EXPERIMENTAL genase [EC 1. 1. 1. 27] (3) and aldose mutarotase [EC] (6) has been reported Materials-Yeast alcohol dehydrogenase was prepar to be reversible and competitive for the sub ed from fresh baker's yeast* by the following pro strate. However, the reactions of yeast and cedures which are essentially based upon the method alcohol dehydrogenases [EC 1. 1. 1. 1] (3), of R a c k e r (11). Eight pounds of fragmented fresh yeast were macerated with 450 ml. of ethyl acetate. yeast ji-fructofuranosidase [EC 3.2. 1.26, pre The mixture was left at room temperature for 40 viously known as ] (7) arid (3-amylase minutes and then centrifuged at 6,000Xg for 30 [EC] (8) were found to be noncompeti minutes. The resulting precipitate was suspended in tivelv inhibited by urea. 4 liters of 0.25 .44 Na2HPO4, and the suspension was

In the presence of higher concentrations adjusted to pH 7.5 with NH4OH, and left at 4°C for of urea, however, Brand, E v e r s e and a week. At the end of this autolysis period, the yeast K a p l a n (4) found that yeast and liver residue was removed by centrifugation. To the alcohol dehydrogenases and various animal supernatant , solid was lactate dehydrogenases lost activity irrever added to 60% saturation. The mixture was left to stand for 30 minutes at room temperature and then sibly and in parallel with the changes in the precipitate was collected by centrifugation, dis their protein structure. On the other hand, solved in cold and dialysed against 0.0211 Harris (5) reported that in the presence Na2HPO4 at 4°C overnight. The dialysed solution of 2.0-8.O M urea trypsin loses reversibly its was quickly brought to 50-55•Ž and maintained at activity in parallel with the unfolding of its this temperature for 15 minutes in a water bath. protein molecule. After cooling, the mixture was centrifuged and the

The action of urea on the various protein Abbreviations used: NAD, nicotinamide adenine molecules has been explained by K a u z man n dinucleotide ; NADH,, reduced nicotinamide adenine * Present address : Department of , dinucleotide ; EDTA, ethylenediamine tetraacetate. ** Baker's yeast was kindly supplied by Oriental College of General Education, University of Tokyo, Meguro-ku, Tokyo. Yeast Co., Tokyo. 73 74 T. OHTA and Y. OGuRA clear supernatant obtained was chilled in a salt-ice of the assay medium was 3.0 ml. The reaction was bath. One-half volume of (-10°C) was added initiated by addition of 0.01 ml. of enzyme solution slowly to the supernatant with continuous stirring and in pyrophosphate buffer. Spectrophotometric readings the mixture was kept at -10°C for 1 hour. The were taken at 10 second intervals and the initial resulting precipitate was removed by centrifugation velocity was determined from the changes in optical at 6,000Xg for 10 minutes or by . Then observed during the first 30 seconds. The the supernatant was again chilled to -10°C, and velocity was expressed as moles of substrate oxidized mixed with an additional 0.55 volume of cold acetone. per second per enzyme unit, assuming that one native The resulting precipitate was collected by centrifuga enzyme molecule contains four enzyme units. tion or filtration, dissolved in 0.01 M phosphate Fluorometry-Fluorometric measurements were buffer, pH 6.5, and dialysed against the same buffer made with a Hitachi EPU-2A spectrophotometer overnight at 4°C. Then the precipitate was removed equipped with a Hitachi G-1 spectrofluorometric by centrifugation and the supernatant was passed attachment which consisted of a grating monochro through a DEAE-cellulose column previously equili meter and a 500 W high pressure xenon arclamp. brated with 0.01 M phosphate buffer, pH 6.5, and The simple was placed in a 1 em.2 quartz cell with washed with an equal volume of the same buffer. low fluorescence and was illuminated with monochro By this treatment nucleic , which disturbed the matic light. The fluorescence emission was measured crystallization of the enzyme, could be completely at right angles to the exciting light beam. Emitted removed. The eluate was then put into cellulose energy was measured by comparison of the observed tubing and left for several hours in 0.35 saturated value with the fluorescence excitation spectrum and ammonium sulfate solution, adjusted to pH 7.5. the absorption spectrum of a dilute solution of fluore Crystals which formed were collected by centrifuga seein in 0.1 N NaOH. The intensity of fluorescence tion and dissolved in 0.01 M phosphate buffer, pH 7.5. was expressed by the quantity F : After four recrystallizations, 200 to 400 mg. of crys talline yeast alcohol dehydrogenase were obtained from 8 1 b of fresh yeast. This enzyme preparation was , where q is proportional to the number of quanta found to be homogeneous by electrophoresis and emitted and T is the transmittancy to the test solution analytical centrifugation, though it was reported by at the wave length of the exciting light. The fluores H a y e s and V e l i c k (13) that two electrophoretically cence emission spectrum of the enzyme was measured different components of yeast alcohol dehydrogenase, using diluted enzyme solution with an optical density; that is, an active component and an inactive de less than 0.2 at 280 mp. naturation product, were present even after recrystalli Ultraviolet Difference Spectrum-The ultraviolet zation. Negligible amount of the denatured component difference spectrum was measured with a Hitachi seems to have been present in our preparation. EPU-2A spectrophotometer. The width of the slit was fixed during any one series of experiments and Urea was purified by the method of B e n e s c h, the same pipettes were employed to reduce pipetting Lard y and Ben e s c h (12), that is, a concentrated errors. The value of the difference in optical density solution of urea was treated with Amberlite MB-1 cation and anion exchange resin at 50-55°C, filtered at any given wave length thus obtained was expressed through a glass filter and crystallized. The NAD by dividing it by that of the reference solution at used was purchased from Sigma Chemicals. NADH2 280 my. was prepared by reduction of the NAD, with yeast Viscometry -Viscosity was measured with an alcohol dehydrogenase and in the presence of Ostwald type viscometer of spiral form. The outflow time for water was 80-100 seconds at 20.0°C. Measure and EDTA, and was purified by ments were carried out in a thermostat maintained repeated alcohol fractionation and washing with . at 20.0°C. The reduced viscosity was defined as follows : The enzyme concentration was determined by measuring the optical density at 280 my, assuming an

extinction coefficient of 1.89 X 105 cm.2 -1 based were r and ƒÅƒÍ are the viscosity of the test solution

on a molecular weight of 150,000 (13 ). and the , and c is the concentration of protein Activity Measurements-Enzymatic activity was de in g. per 100 ml. termined by measuring the increase in optical density Sedimentation Analysis-Sedimentation measurments at 340 my due to formation of NADH,. The measure were carried out with a Spinco model E analytical ments were made at pH 8.5 and 20°C using a ultracentrifuge. The sedimentation patterns were Hitachi EPU-2A spectrophotometer. The total volume obtained by schlieren optics at a rotor speed of 59,780 Effects of Urea on Yeast Alcohol Dehydrogenase 75 r.pm, and temperatures between 10° and 28°C re urea, but the rate of the reaction decreased gulated by the automatic temperature control unit. with increase in the concentration of urea. The molecular weight of the enzyme was de The enzyme was not preincubated with urea, termined with an ultracentrifuge by Y p h a n t i s' but there was no lag in the inhibition. This procedure (14). 'Experiments were carried out using shows that the inhibitory effect of urea is a cell with seven observation channels at a rotor instantaneous. speed of 12,590 r. p. m. at 15°C. The initial concen tration of protein was determined by numerical in Effect of Dilution on Enzyme Stability-The tegration of the synthetic boundary trace. effect of the concentration of enzyme on its stability with urea was tested. Enzyme solu

RESULTS tion of various concentrations was incubated in 1.0 M urea at pH 8.5 and 20°C for 10 Part 1. Kinetic Analysis of the Yeast minutes before assays. The concentration Alcohol Dehydrogenase Reaction of enzyme tested ranged from 0.02 to 0.06 mg.

Time Course of Reaction in the Presence and per ml. Assays were made in the presence

Absence of Urea-The reaction was initiated by of LO M urea. The same concentration of

introducing 0.01 ml. of buffered enzyme solu enzyme in the different assay media was

tion into assay media containing various con obtained by adding different volumes of en

centration of urea. As shown in Fig. 1, the zyme solution. Fig. 2 shows that when the

optical density at 340 mƒÊ increased linearly concentration of enzyme in the incubation

with reaction time even in the presence of medium was less than 0.05 mg. per ml., the

F o. 2. Effect of dilution of enzyme on its stability in the presence of 1.0 M urea. Various concentrations of enzyme in pyro

FIG. 1. Time course of reduction of NAD phosphate buffer (0.01 M, pH 8.5) were incubated at 20°C for 10 minutes in the presence of M urea. in presence and absence of urea. The assay was made at pH 8.5 and 20°C by in Assay mixtures contained enzyme (5.ZX troducing this preincubated enzyme into the 10-9M), pyrophosphate buffer (0.02M, pH 8.5), reaction mixtures. Assay mixtures contained 3.3 ethanol (0.1 M), NAD (8.8 X 10-4 M), ammonium X 10-9 M enzyme, 0.67 mM NAD, 0.1 M ethanol sulfate (ca. 0.3 mM) and various concentrations and 1.0 M urea. Ordinate : rate of reaction per of urea. The urea concentrations used were enzyme unit. Abscissa : concentration of enzyme none (0), 1 M (!) and 2 M (x). Temperature : in the incubation mixture. 20°C. 76 T. OHTA and Y. OGURA activity per mole of enzyme unit decreased activity was studied. Enzyme was preincubat to zero, whereas the activity was almost ed with 1.0 or 2.0 M urea solution at pH 8.5 independent of the concentration of enzyme and 20°C. At intervals, aliquots of the in when higher enzyme concentration was used cubated mixture were taken out and their during the preincubation. The stability of activity was measured in of the same the enzyme seemed to increase with the ionic urea concentration as those in the incubation strength of the incubation mixture. mixture. As shown in Fig. 3, the remaining Although the reason for the instability of activity was not affected by the incubation dilute solution of enzyme is not clear, it may time. Even after a two hour incubation be due to slight heavy metal contamination period with 2.O M urea, the activity remained of the urea sample used (15, 19) or to the was found to decrease very little. enzyme protein developing a labile structure Reversibility and Irreversivility of Urea In at high dilution. The enzyme concentration hibition--The enzyme was incubated at pH 8.5 in the incubation mixture was more than and 20°C for 10 minutes in the presence of 0.05 mg. per ml. in the experiments described urea at various concentrations, and then the below. activity was assayed by introducing 0.01 ml. Effect of Preincubation with Urea-The effect aliquots of these solutions into 3 ml. of reac of preincubation with urea on the enzymatic tion mixture. The urea was diluted three hundred times in the assay medium by this procedure, and tests showed that such low concentrations of urea had no inhibitory effect upon the enzyme reaction. The activity was recovered im mediately on dilution of the urea and no induction period was observed even in the initial part of the reaction. Fig. 4 shows that the effect of urea was completely reversible when the urea concentration was less than 2.OM and that it was irreversible above 2.OM urea. Thus, when the enzyme had been incubated with 4.5M urea its activity could not be recovered after removal of the urea. The data obtained are in good accor dance with those reported by B r a n d, E v e r s e and Kalpan (4). However, Sun d (.16) reported that degree

FIG. 3. Stability of yeast alcohol dehydrogen of inhibition of the yeast alcohol dehydroge ate against urea. nase reaction by urea varies with the incuba Enzyme (0.097 mg. per ml.) was incubated tion time. In his experiments, activity was with urea (-•-, 1 M; -0-, 2 M) in pyro completely lost by treatment of 2.0 M buffer (0.1 M, pH 8.5) at 20°C. Incu for 10 minutes and did not recover even after bation mixtures contained ca. 30 mM ammonium removal of the urea. Thus his results do not sulfate. Assay was carried out at 20°C and pH 8.5 agree with those given in Fig. 4. This dis in the presence of urea at the same concentrations crepancy is presumably due to differences in as those in the preincubation media. Assay mix tures contained 2.1 X 10-9 M enzyme, 0.1 M etha the enzyme concentration used or the ionic nol, 0.68 mM NAD, 0.1 M pyrophosphate buffer strength of the incubation mixtures, since the (pH 8.5) and urea (*, 1 M; 0, 2 M). Ordinate : enzyme stability against urea decreased on rate of reaction per enzyme unit. Abscissa : time dilution of the enzyme, as shown in Fig. 2, of incubation with urea. and also on lowering the ionic strength of Effects of Urea on Yeast Alcohol Dehydrogenase 77

the following formula :

The value of Kr, which is the urea concent ration at 50% inhibition, was found to he 1.1 M at pH 8.5 and 20°C. Similar experiments to those described above were made using other substrates such as propanol, isopropanol and glycol. The relationships obtained were also represent ed by formula (1) and the value of Ku was found to be 1.1 M with all the substrates tested.

Fin. 4. Effect of urea concentration on activity of yeast alcohol dehydrogenase. For results indicated by open circles, the reaction was initiated by introducing buffered enzyme solution into assay mixtures containing various concentrations of urea. Enzyme was not preincubated with urea. For results indicated by solid circles, enzyme was preincubated at pH 8.5 and 20°C with the various concentrations of urea indicated on the abscissa and then introduced into assay mixtures containing no urea. The incubation mixtures contained 1.56µM enzyme, 0.1 M pyrophosphate, ca. 30 mM ammonium sul Fro. 5. Effects of various concentrations of fate and various concentrations of urea. In both urea or its analogues on the activity of yeast experiments assay with or without urea was made alcohol dehydrogenase. Assay mixtures contained in 0.1 M pyrophosphate buffer at 20°C and pH 8.5 enzyme (1.8 X 10-9 M), pyrophosphate buffer in the presence of 5.2 x 10-9 M enzyme, 0.88 mM (0.01M, pH 8.5), NAD (4.Ox 10_4 M), ethanol NAD and 0.1 M ethanol. (0.1 M), ammonium sulfate (ca. 0.3 mM) and urea or its analogues. -0-, urea; -•~-, forma

incubation mixture*. mide; -ƒ¢-, ; -•-, . Assay Relationship between Relative Activity and was made at 20°C. Enzyme was not preincubated Concentratiunof Urea-Activities were measured with urea. Ordinate : percentage of relative ac in the presence of various concentrations of tivity. Abscissa : concentration of urea or urea urea and constant concentrations of ethanol analogue on logarithmic scale. and NAD. The data indicated by open Effects of Urea upon the Michaelis Constant circles in Fig. 5 show the relationship between and the Maximum Velocity.-First the Michaelis the relative activity (vi/v) and the logarithm constant and the maximum velocity of native of the urea concentration [U], where vi and yeast alcohol dehydrogenase were measured v represent the rates of the reaction in the at pH 8.5 and 20°C in the absence of urea. presence and absence of urea, respectively. The measurements were made using ethanol The relationship obtained is represented by and isopropanol as substrates in the presence

* The detail will be reported in the next paper. of given concentrations of NAD. Figs. 6 and 7 show the Lineweaver-Burk plots obtained The experiment shown in Fig. 4 was carried out using ethanol and isopropanol. On plotting using an incubation mixture of a rather high ionic strength. the reciprocals of the apparent maximum 78 T. OHTA and Y. OGURA

velocities (Vap) against those of the concentra constants of neither the acceptor tions of NAD, a linear relationship was nor the donor. were affected by the addition obtained. The values* of the true maximum of urea. However, the maximum velocity velocity (Vm), which should be obtained at infinite concentrations of both hydrogen acceptor and donor, were found to be 460 sec."' (ethanol) and 33 sec.-' (isopropanol). The results shown in these figures indicate that the NAD concentration does not affect the Michaelis constants for and vice versa. The Michaelis constants were found to be 1.67x10-2'M (ethanol), 0.18M (isopropanol), 7.15 x 10-4 M (NAD) and 7.7 x 10-5M (NADH2), respectively. These results are in good agree FIG. 7. Lineweaver-Burk plot of yeast alcohol ment with those obtained by H a y e s and dehydrogenase reaction. Isopropanol was used as Velick (13). substrate. Assay mixtures contained enzyme (4.45 To elucidate the mode of inhibition of X 10-8 M), pyrophosphate buffer (0.02 M, pH 8.5), urea, kinetical experiments similar to those NAD (-•ü-,7.67x10-4M; -ƒ¢-,3.83•~10-4M; -•œ-, l described above were performed in the .15 X 10-4 M), ammonium sulfate (ca. 0.3 mM) and isopropanol. Isopropanol concentrations presence of urea less than 2.0 M. Fig. 8 shows of 0.067 to 0.3 M were used. Temperature : 25°C. the Lineweaver-Burk plots for ethanol with or v/e : Rate of reaction per enzyme unit. Vap/e : without urea. The data obtained are in Apparent maximum velocity per enzyme unit at accord with those of R a j a g o p a l a n et al. given concentration of NAD. (3). The Lineweaver-Burk plots for NAD and NADH2 were also studied in the presence of urea. As shown in Table I, the Michaelis

FIG. 6. Lineweaver-Burk plot of yeast alcohol dehydrogenase reaction. Ethanol was used as

substrate. Assay mixtures contained enzyme (1.44

x10-9M) pyrophosphate buffer (0.02M, pH8.5), FIG. 8. Lineweaver-Burk plots for ethanol in

NAD (-0-, 7.67 x 10-4M; -L --, 3.83 x 10-4M; presence and absence of urea. Assay mixtures -•œ-, 1.15•~ l0-4M), ammonium sulfate (ca . contained enzyme (2.11•~10-9M) pyrophosphate 0.3mM) and ethanol. Concentrations of ethanol buffer (0.02 M, pH 8.5), NAD (8.9x 10-4 M), of 0.01 to 0.1 .19 were used. Temperature : 25°C. ethanol (0.01 to 0.1 M), ammonium sulfate (ca. 0.3 v/c : Rate of reaction per enzyme unit. Vap/e : mM) and urea (-0-, none; -ƒ¢-, 1 M; -•œ-,

Apparent maximum velocity per enzyme unit at 2 M). Assay was made by introducing buffered

given concentration of NAD. enzyme solution into the reaction mixture. Enzyme was not preincubated with urea. Tem * The values obtained were those of the fresh perature : 20°C. v/e : Rate of reaction per .,enzyme preparations. enzyme unit. Effects of Urea on Yeast Alcohol Dehydrogenase 79

TABLE I Effect of Urea Concentration on Kinetic Values of Yeast Alcohol Dehydrogenase

The concentration of enzyme was 2.11 •~ 10-s M. Assay was made in 0.02 M pyrophosphate buffer at

pH 8.5 and 20•Ž.

TABLE II TABLE III Effect of Dielectric Constant on Activity Effect of Viscosity of Solution on Activity of Yeast Alcohol Dehydrogenase of Yeast Alcohol Dehydrogenase

1) Values were quoted from the International 1) Values were quoted from the International Critical Tables Critical Tables. Assay mixtures contained : enzyme (1.8 The experimental conditions were the same •~10-9 M), pyrophosphate buffer (0.01 M, pH 8.5), as those described in Table II. The control, NAD (4.0 •~104 M), ethanol (0.1 M) and urea or assay was made in the absence of urea and sucrose. . The control, assay was made in the absence of urea and glycine. Temperature : 20•Ž investigated. Fig. 5 shows the relationships

( Vm) decreased markedly with increase in the between the relative activities and the logari

concentration of inhibitor. Similar results thms of the concentrations of the analogues.

were obtained with propanol or isopropanol All the relationships obtained conformed to

as the substrate. From these results, it is formula (1) given above. The inhibitions

suggested that the inhibition caused by urea by these analogues were noncompetitive and

is noncompetitive for either the hydrogen reversible. The findings seem to indicate

donor or the acceptor under the present con that these analogues act on the enzyme

ditions. molecule in the same way as urea does. Analogues of Urea-The inhibitory effects However, ionic analogues of urea, such as

of non-ionic analogues of urea, such as salts, inhibit the enzyme more

formamide, acetamide and thiourea on the strongly and the sigmoid inhibition curves obtained were of higher order than that of yeast alcohol dehydrogenase reaction were also 80 T. OHTA and Y. OGURA urea. presence of urea at various concentrations, Since addition of urea increased the assuming that the increment of fluorescence viscosity and dielectric constant of the medium, intensity was proportional to the amount of the effects of increase in viscosity and di NADH2 attached to the enzyme molecule. electric constant caused by addition of sub Fig. 9 shows that below 2.0 M urea the fluo stances other than urea on the enzyme activity rescence intensity slightly increased*, but the were studied. The viscosity and dielectric fluorescence decreased markedly at higher constant of the medium were increased by concentrations of urea. From these results, addition of sucrose or glycine instead of urea. the amount of bound NADH2 does not seem The results, shown in Tables II and III, to be affected appreciably by addition of urea indicated that the inhibition caused by urea despite the marked decrease in enzyme activity is probably not due to increase in the viscosity in the presence of 0.5-2.0 M urea. This or dielectric constant of the solution. accords with the kinetic data showing that the Effect of Urea on the Fluorescence of the Michaelis constant of NADH2 was not affected NADH2-Enzyme Complex-It is known that by urea under the conditions mentioned above. enhancement of the fluorescence emission and The data also suggest that at urea concentra a shift in the maximum of the emission spe tions above 2.0 M, the amount of bound ctrum of NADH2 are induced by addition of NADH2 decreases markedly with increase in the enzyme (17). Since this indicates the concentration of urea and finally that the formation of an NADH2-enzyme complex, the binding of NADH2 with enzyme molecule effects of urea upon the dissociation constant does not occur in the presence of urea above of the NADH2-enzyme complex were studied 4.0 M. This finding is of interest in com by measuring the intensity of fluorescence at parison with the curve in Fig. 4 which shows 430 mƒÊ induced by exciting a test solution the reversible and irreversible loss of the with a monochromatic light of 340 m s in the activity. Effect of on Enzymatic Activity -Effect of thiocyanate upon the yeast alcohol dehydrogenase reaction was investigated kine tically to compare its inhibition with that of urea, since thiocyanate is known to act as a protein denaturant. It was found that concentrations of thiocyanate less than 0.3 M inhibit the enzyme reaction reversibly, while at higher concentrations the inhibition is irre versible (Fig. 10). Kinetic experiments similar to those with urea were carried out at lower concentrations of thiocyanate. Fig. 11 shows the Lineweaver-Burk plots for the ethanol concentration obtained with and without 0.05

* Under these conditions , this slight increase in the fluorescence intensity may be caused by a slight FIG. 9. Effect of urea concentration upon increase in the amount of bound NADH2 or by a fluorescence intensity of NADH2-enzyme com solvent effect induced on addition of urea. On the

plex. Test solution contained enzyme (8.54•~ other hand, Brand, Everse and Kaplan (4) 10-6m), NADH2 (1.03 x l0-5 M ), pyrophosphate found that in the presence of urea there is a marked buffer (0.03M, pH 8.5), ammonium sulfate (ca. increase in the fluorescence of NADH2 when it is

30 mM) and urea. Abscissa : urea concentration. bound to horse liver alcohol dehydrogenase, though Ordinate : fluorescence intensities at 430 m/s. not when bound to the enzyme. They suggested that Excitation was made by monochromatic light at urea forms a ternary complex with NADH2 and the

340 m/I. Temperature : 20°C. enzyme. Effects of Urea on Yeast Alcohol Dehydrogenase 81

M thiocyanate in the presence of 0.81 x 10-1 M NAD. Fig. 12 shows similar plots for the NAD concentration in the presence of 0.1 M ethanol with and without 0.05 M thiocyanate. The findings indicate that the inhibition by thio , unlike that by urea, is competitive for ethanol, but noncompetitive for NAD. The relationship between the thiocyanate concentration and enzymatic activity was in vestigated in the presence of constant con centrations of ethanol and NAD. The data

FIG. 11. Lineweaver-Burk plot for ethanol in the presence and absence of . Assay mixtures contained enzyme (1.43 x l0-9 M ), pyrophosphate buffer (0.02 M, pH 8.5), NAD (8.1 x 10-4 M), ethanol (0.01 to 0.1 M) and potassium thiocyanate (-0-, none; -•--, 0.05M). The reaction was initiated by introducing buffered enzyme solution into the assay medium and the enzyme was not preincubated with thiocyanate. Temperature : 20°C. v/e : Rate of reaction per enzyme unit.

FTC. 10. Effect of thiocyanate concentration on enzyme activity. For results indicated by broken line, assay was made in the presence of potassium thiocyanate (X). Reactions were initi ated by introducing buffered enzyme solutions into assay mixtures containing various concentra tions of potassium thiocyanate. Enzyme was not preincubated with thiocyanate. Assay media con tained 0.74 mM NAD, 0.1 M ethanol, 3.4 X 10-s M enzyme, 0.02M pyrophosphate (pH 8.5), ammo nium sulfate (ca. 0.3 m

minutes. There was a remarkable difference in the optical density between 270 and 300 m,a, where two troughs at 280 and 286 mp and a small shoulder at 292 mp were observed. Although this difference spectrum obtained can be considered to be overlapped spectra due to the changes of the environments of both tyrosyl and tryptophyl residues, the resulting changes of the spectrum at 280 and 286 mp may be mainly assigned to that in the environment of the tyrosyl residues of the enzyme protein, since the contribution of the tryptophyl residues can be presumed to appear at 290-295 mp. The shoulder at 292 mp may FIG. 13. Relationship between reciprocal of be attributed to the tryptophyl residues in the relative activity and thiocyanate concentration. enzyme molecule. The change in the environ Assay mixtures contained enzyme (1.07 X 10-9 M), ment of tyrosyl and tryptophyl residues may pyrophosphate buffer (0.02 M, pH 8.5), NAD (7.4 be caused by an unfolding of the enzyme X 10-4 M), ethanol (0.1 M) and various concent rations of potassium thiocyanate. Enzyme was molecule (22). The relationship between the not preincubated with thiocyanate. v : Activity change in the spectrum of the enzyme protein in absence of thiocyanate. vi : Activity in presence and the concentration of urea added was of thiocyanate. Temperature : 20°C. further investigated by plotting the changes in optical density at 286 my against the concen tration of urea. The data obtained are given obtained are shown in Fig. 13. The plot of in Fig. 15. When the concentration of urea the reciprocals of the relative activities against the thiocyanate concentration gave a straight added was higher than 2.0 M, the optical density decreased markedly with increase in line through unity on the ordinate. The in concentration of urea and approached a hibition constant, Ki, for thiocyanate was minimum value at 4.0 M urea. However, the found to be 6 X 10-3 M at pH 8.5 and 20°C optical density did not decrease at concentra assuming a value of 1.7 X 10-2 M as the tions of urea less than 2.0 M, but instead Michaelis constant for ethanol. increased slightly. This increase seems to have Part 2. Analysis of the Structural Changes been caused by an increase in the refractive in the Enzyme Protein Molecule index of the medium. It seems of interest to compare the data given in Fig. 15 with the Ultraviolet Difference Spectrum-The ultra kinetic data obtained previously. No unfold violet absorption spectrum of the enzyme was ing of the protein molecule occured on in investigated in the presence and absence of cubation with a low concentration of urea urea to see whether any conformational with which reversible inhibition was observed. change of the enzyme protein was related to Between 2.0 and 4.OM urea, the percentage the reversible or irreversible loss of activity of unfolding of the molecule* was roughly induced by urea. Fig. 14 shows the difference proportional to the irreversible loss of activity spectrum of enzyme solutions with and without observed. urea in the ultraviolet region. In this experi Fluorescence Emission Spectrum-The fluores ment, enzyme solution containing 8.0 M urea cence emission spectrum of the enzyme was was placed in the test cell and the same

concentration of enzyme without urea in the * It was assumed that the concentration of the reference cell. Spectrophotometric measure native enzyme molecure can be expressed as a linear ments were made after the enzyme solution function of 0. D.286 according to Beer's law in the had been preincubated with urea for 10 presence of various concentrations of urea. Effects of Urea on Yeast Alcohol Dehydrogenase 83

FIG. 15. Effect of urea concentration on the optical density at 286 my. The test cell contained FIG. 14. Ultraviolet difference spectrum of yeast enzyme (6.21 X 10-6 M), pyrophosphate buffer alcohol dehydrogenase in the presence and absence (0.022 111,pH 8.5), ammonium sulfate (ca. 50 mM) of 8 M urea. The test cell contained enzyme and urea at the concentrations indicated. The ,(6.21•~10-6M), pyrophosphate buffer (0.022141, reference cell contained all the same components pH 8.5), ammonium sulfate (ca. 50 mM) and except urea. Measurements were made after in urea (8 M). The reference cell contained all the cubation for 10 minutes. Temperature : 20°C. same components except urea. Temperature : LO.D.286 represents. 20°C.

measured by exciting dilute solutions of the attributed to the fluorescence of the tyrosyl enzyme with monochromatic light of 280 mp residues in the enzyme protein. The shoulder after preincubating them for 10 minutes with observed between 330 and 340 mp shifted to and without urea. Fig. 16 shows that the longer wavelengths with increase in urea con fluorescence spectrum of the native enzyme centration and appeared as a distinct peak has a maximum at 310 mp and a shoulder in the presence of 8.0 M urea. The second between 330 and 340 m,u, and that consider peak of the denatured enzyme observed at able changes in the spectrum were observed 345 mp may be attributed to the fluorescence on addition of urea. That is, the fluorescence of the tryptophyl residues in the enzyme intensity at 310 ma decreased markedly with protein. Fig. 17 shows the plot of the fluores increase in concentration of urea. W e b e r cence intensity at 310 ma against the concent

(23) reported that the fluorescence of the ration of urea. A marked decrease in fluores tyrosyl residues of many is quenched cence intensity was observed in the range and only that of their tryptophyl residues between 2.0 and 4.0 M urea. This relation ,can be observed . On the other hand, K o n e v ship seems to agree with the relationship

(24) mentioned that the fluorescence spectra between the ultraviolet absorption spectrum of some proteins, which have an emission and the concentration of urea. Thus , the maximum at 313 mp, may result from their data seem to confirm the previous results, that tyrosyl residues. Therefore, the emission peak a change in the environment of the tyrosyl of the enzyme observed at 310 mƒÊ may be residue caused by an unfolding of the protein 84 T. OHTA and Y. OGURA

FIG. 17. Effect of urea concentration on fluorescence intensity. Fluorescence intensities were measured at 310 mp. Excitation was made by monochromatic light at 280 m)C. Measure ments were made at 20°C in the presence of FIG. 16. Fluorescence emission spectrum of various concentrations of urea under the condi

the enzyme in the presence of various concentra tions described in Fig. 16. tions of urea. Samples contained enzyme (7.90•~ 10-7 M), pyrophosphate buffer (0.011 M, pH 8.5), viscosity above 2.OM urea may be attributed. ammonium sulfate (ca. 10mM) and urea at the to an increase in the effective hydrodynamic concentrations indicated. The numbers represent volume of the enzyme molecule. This suggests the concentration (M) of urea added. Try ; emis that a large unfolding of the peptide chainss sion spectrum of of tryptophan. in the enzyme molecule occurs together with Excitation was made by monochromatic light at a swelling of the molecule in the presence of 280 mp. Temperature : 20°C. urea more than 2.0 M. Thus, the result seems molecule may be induced by the addition of to be compatible with those obtained previously sufficient amount of urea. by spectrophotometry in the ultraviolet region Viscosity Measurements-The viscosity was and by fluorometry. U 1 m e r and V a 11 e e measured to confirm the results obtained by (25) found that denaturation of yeast alcohol optical methods. Prior to the measurements, dehydrogenase caused by urea leads to, enzyme solution was dialysed against 0.1 M simultaneous changes in the specific rotation pyrophosphate buffer, pH 8.5, at 4°C for 20 of the protein and the dispersion constant, hours, and then centrifuged at 100,000x g for and that the magnitude of the change descri one hour. The supernate was diluted with bed above is proportional to the irreversible dialyzate to an appropriate concentration just loss of activity. Brand, Everse and before the measurements. After 10 minutes K a p 1 a n (4) have also reported that the to attain thermal equilibrium, the viscosity of changes in optical rotation at 435.5 mo and the test solution was measured using an protein fluorescence of the enzyme caused by Ostwald viscometer at various concentrations urea were proportional to the irreversible loss of urea. The data obtained are illustrated in of activity. Their data seem to be in good Fig. 18. The reduced viscosity of yeast alcohol agreement with those reported here. dehydrogenase increased markedly above 2.0 M Sedimentation Analysis-The enzyme solu urea, but increased only slightly below 2.0 M tion was dialysed against 0.1 M pyrophosphate urea. The marked increase in the reduced buffer, pH 8.5, at 4°C for 20 hours, and then Effects of Urea on Yeast Alcohol Dehydrogenase 85

value of the second peak in the presence of 2.O M urea. These findings indicate that the disintegration of native molecule into small subunits with an s20, ,U value of 2.5 takes place below 2.O M urea. Since the inhibition caused by a concent ration of urea less than 2.0 M was found kinetically to be reversible, tests were made of whether the dissociation of the enzyme molecule mentioned above was reversible, that is, whether the 7.0 S component could be reformed from the subunits of 2.5 S when the urea was removed. Enzyme solution was incubated with 2.0 M urea for 10 minutes and then divided into two parts. One was diluted with buffer solution to a urea con FIG. 18. Effect of urea on viscosity of yeast centration of 0.5 M and the other was diluted alcohol dehydrogenase. Test solutions contained with 2.0 M urea solution so that the protein enzyme (3.05 g. per 100 ml.), pyrophosphate buffer* (0.033M, pH 8.5) and various concentra concentrations of the two were identical. The tions of urea. Measurements were carried out at data obtained by sedimentation analysis are 20.0°C. shown in Figs. 19 C and D. The former * By comparison with a solution containing sample, containing 0.5 M urea, showed a quite similar sedimentation pattern to that obtained 0.1 M chloride, it was found that no electroviscous effect was observed at this con in the presence of 0.5 M urea without prior centration of buffer, since the addition of sodium incubation with 2.0 M urea. However, 7.0 S chloride had no effect on the reduced viscosity component was scarcely observed in the sedi of the enzyme solution. mentation pattern of the latter sample. This indicates that reformation of 7.0 S component from the dissociated subunits probably occurs centrifuged at 100,000xg for one hour and on removal of the urea. However, when diluted to 5.0 mg. per ml. with the same enzyme solution was preincubated with 4.5M buffer or buffer containing an appropriate urea, the peak of the 7.O S component did amount of urea. The solutions thus treated not appear, as may be seen in Fig. 19 E, were subjected to sedimentation analysis using even when the urea concentration in the test a Spinco ultracentrifuge type E. The data solution was diluted to 0.5 M before sedimen obtained are shown in Fig. 19. The native tation analysis. This finding agrees with the protein showed a.sedimentation pattern with evidence obtained previously that the activity a single peak and the value of the sedimen of the enzyme was lost on incubation with tation constant, s20,u,, was found to be 7.0 5, as 4.5M urea even after a subsequent removal reported by other authors (19, 26, 27 ). When of the urea from the test solution. 1.0 M urea was added to the test solution The effect of thiocyanate on the sedimen immediately before sedimentation analysis, a tation pattern was also investigated for com second, more slowly moving peak with s20,w parison with the evidence inferred from kinetic value of 2.5 appeared besides the peak at data. On the addition of 0.04 M thiocyanate 7.0 S mentioned above (16). On increasing to the assay medium a marked inhibition* the urea concentration, the amount of the could be presumed from the kinetic results. 2.5 S component increased with concomitant decrease in that of the native 7.0 S component * Since thiocyanate is a competitive inhibitor and finally the amount of the latter became for. the substrate, the-percent inhibition was computed almost negligible without affecting the s20,u, at a very low concentration of substrate. 86 T. OHTA and Y. OCURA

2 M urea. D (below) : pattern of the enzyme (5 mg. per ml.) in 0.5 M urea without preincu bation with urea. E (above) : pattern of enzyme (3.6 mg. per ml.) in 0.5 M urea after incubation for 10 minutes with 4.5 M urea. E (below) : pattern of enzyme (5 mg. per ml.) in the absence of urea.

However, no decrease in the amount of the 7.0 S component was detected in the sedimen tation pattern under these conditions. These findings indicate that no dissociation of the enzyme molecule occurs in the presence of 0.04M thiocyanate, although thiocyanate is known to be a protein denaturant. Determinationof the Molecular Weight-Since it is known that the native molecule of yeast alcohol dehydrogenase is composed of four subunits and can dissociate into its subunits by exposure to chelating agents (26) or sodium dodecyl sulfate (27), it is of interest to see how many fragments can be produced from one native enzyme molecule by the dissocia tion reaction induced by addition of urea. The molecular weights of the native enzyme molecule and the dissociated subunits having an s20, u, value of 2.5 were determined by Yphantis' method. Prior to the measure ments, the protein solution was dialysed against buffer, as described previously. Measurements were made at pH 8.5 with and without urea using this dialysed enzyme (6.5 mg. per ml.). Assuming 0.769 cm.3/g. as the value of the partial specific volume of the enzyme protein molecule (13), the molecular weight was calculated from the data obtained 195 minutes after maximum speed was attained FIG. 19. Sedimentation patterns of yeast (12,590 r. p. m.). The molecular weight of the alcohol dehydrogenase in urea solution. Photo native enzyme was found to be 14.6 x 104,which graphs were taken 100 minutes after addition of is in good agreement with that reported by urea. The speed was 59,780 r. p. m. A (above) : other authors (13, 26, 27), and the average pattern of the enzyme (5 mg. per ml.) in 2 M urea molecular weight of the mixture of the native solution. A (below): pattern of enzyme (5 mg. enzyme and dissociated fragments was found per ml.) in the absence of urea. B : pattern of to be 7.85 x 104 in the presence of 1.0 M urea. enzyme (5 mg. per ml.) in M urea solution. C The faster moving component in this sample (above) : pattern of the enzyme (5 mg. per ml.) in was estimated by measurement of the sedi 2 M urea solution after incubation for 10 minutes mentation pattern to constitute 38% of the with 2 M urea. C (below): pattern of the enzyme total*. Therefore, the molecular weight of (5 mg. per ml.) in 0.5 M urea solution after incu bation for 10 minutes with 2 M urea. D (above) : * This value was calculated from the appa rent pattern of the enzyme (5 mg. per ml.) in 0.5 M ratio of the two components. However, no correction urea solution after incubation for 10 minutes with for the j o h n s t o n-O g s t o n effect (34) was performed . Effects of Urea on Yeast Alcohol Dehydrogenase 87 the slower moving component was estimated scheme (31) : a ternary complex, coenzyme to be 3.7 X 104 by the following calculation: substrate-enzyme-compound, can be formed, without interaction between coenzyme and substrate, by the independent binding reac tions of both reactants with the enzyme On the other hand, K a g i and V a l l e e molecule and the oxidation and reduction (26) observed a dissociation of native yeast reactions occur through the active ternary alcohol dehydrogenase into four 2.8 S com complex. In addition, the Michaelis constants ponents on treatment with chelating agents, for both reactions may be very close to the such as 1, 10-phenanthroline or 8-hydroxy dissociation constants of the complexes be quinoline-5-. They estimated the tween the reactants and the enzyme. H aye s molecular weight of these fragments to be and V e l i c k (13) confirmed this by direct 36.000. H e r s h (27) obtained the same result measurement of the amount of coenzyme at using sodium dodecyl sulfate. This value is tached to the enzyme. in good agreement with that obtained for the The formula (2) described above is in 2.5 S component of our sample. Since the agreement with that obtained by N e g el e i n sedimentation pattern obtained in the presence and Wulff (32) and Hayes and Velick of 0.5 M urea had two peaks, as described ( 13) at pH 7.9 but not with the results previously, it is suggested by G i 1 b e r is theory obtained by N y g a a r d and T h e o r e l l (33) (28), that the native tetrameric enzyme mole at pH 7.15 and 6.0. This discrepancy seems cule can be dissociated into four monomeric to be due to differences in pH of the media subunits in the presence of urea, but not into used. two dimeric fragments nor into one monomeric Since the relationship between the urea and one trimeric fragment. concentration and relative activity is expressed by the equation (1 ), the rate of the reaction DISCUSSION in the presence of urea (v,) can be represented It is known that yeast alcohol dehydroge by the following equation : nase combines with 4 moles of coenzyme and contains 4 gram atoms of zinc in an enzyme molecule* (29). Thus, it is assumed that one enzyme molecule has 4 "active centers" From the results, it is suggested that there is which are able to catalyse the oxidation of a strong interaction in the binding reaction alcohol and the reduction of NAD and the with urea and enzyme, that is, almost two reverse reaction. Since Lineweaver-Burk plots molecules of urea combine simultaneously for the substrate as well as for the coenzyme with specific sites on a single enzyme unit which differ from the substrate and coenzyme give straight lines in all cases, the 4 " active centers" must behave independently during binding sites. Since thiocyanate reversibly inhibits the the reaction. From the results obtained the rate of the enzyme reaction (v) in the absence enzyme reaction by competing with the sub of urea may be expressed by the following strate for substrate binding site on the equation : enzyme when it was used as inhibitor, the following equation seems to represent the results obtained from kinetic analysis:

where [s] represents the concentration of sub strate and [c) that of coenzyme. Equation were Ki is the inhibition constant of thiocya (2) was derived from the following reaction nate and [I) the concentration of thiocyanate. * However, W a 11 e n f e l s et al. (31) reported Thiocyanate seems to combine with a posi tively charged group which is involved in the that ca. 5 gram atoms of zinc may be present in the substrate binding site. yeast alcohol dehydrogenase molecule. 88 T. OHTA and Y . OGURA

The findings from physicochemical analy (2) Hill, R. L., Schwartz, H. C., and Smith, E. L., sis suggest that addition of urea causes the J. Biol. Chem., 234, 572 (1959) reversible dissociation of the enzyme molecule (3) Rajagopalan, K. V., Fridovich, I., and Handler, into four subunits, and that these dissociated P., J. Biol. Chem., 236, 1059 (1961) subunits have practically the same folded (4) Brand, L., Everse, J., and Kaplan, N.O., Bio structure as that of the subunits in the native chemistry, 1, 423 (1962) enzyme molecule, provided that the urea con (5) Harris, J. I., Nature, 177, 471 (1956) (6) Chase, A. M., Lapedes, S. L., and von Meier, centration is low. These subunits still seem H. C., J. Cellular and Comp. Physiol., 61, 181 to have essential zinc atoms one for each, (1963) since the active tetrameric enzyme molecule (7) Chase, A. M., von Meier, H. C., and Menna, is reformed from the dissociated subunits on V.J., J. Cellular and Comp. Physiol., 59, 1 (1962) removal of urea. Further addition of urea (8) Weintraub, B.D., Hamilton, G.A., Henshaw, C., apparently causes irreversible conformational and Chase, A. M., Arch. Biochem. Biophys., 107, change in the split subunits, which may cause 224 (1964) loss of their capacity to bind with coenzyme (9) Kauzmann, W., " The Mechanism of Enzyme and which causes irreversible loss of enzyme Action ", ed. by W. D. McElroy and B. Glass, activity. However, further investigation seems Johns Hopkins press, Baltimore, p. 70 (1954) to be necessary to decide whether the rever (10) Klotz, I. M., Science, 128. 815 (1958) sible and noncompetitive inhibition of urea (11) Racker, E., J. Biol. Chem., 184, 313 (1950) (12) Benesch, R. E., Lardy, H. A., and Benesch, R., observed by the kinetic method is caused by J. Biol. Chem. 216, 663 (1955) formation of an inactive enzyme complex (13) Hayes, J.E. JR., and Velick, S.F., J. Biol. Chem,, produced by binding of the urea molecule 207, 225 (1954) or by dissociation of the native enzyme (14) Yphantis, D. A., Ann. N. Y. Acad. Sci., 88, 586 molecule into four subunits, though both (1960) reversible inhibition and splitting of the native (15) Hoch, F. L., Martin, R. G., Wacker, W. E. C., molecule were observed at concentrations of and Vallee, B. L., Arch. Biochem. Biophys., 91, urea less than 2.0 Al. 166 (1960) (16) Sund, H., Biochem. Z., 333, 205 (1960) SUMMARY (17) Duysens, L. N. M., and Kronenberg, G. H. M., Biochem. et Biophys. Acta, 26, 437 (1957) The action of yeast alcohol dehydrogenase (18) Wallenfels, K., and Sund, H., Biochem. Z., 329, was inhibited reversibly by the presence of 17 (1957) urea less than 2 M. From kinetic data, it (19) Snodgrass, P.J., Vallee, B, L., and Hoch, F.L., was assumed that urea does not compete with J. Biol. Chem., 235, 504 (1960) either substrate or coenzyme. The results of (20) Hoch, F. L., and Vallee, B. L., J. Biol. Chem., sedimentation analysis suggested that the 221, 491 (1956) reversible dissociation of the enzyme molecule (21) Redetzki, H. E., and Nowinski, W. W., Nature, into four subunits was caused by the presence 179, 1018 (1957) of urea less than 2M. The enzymatic reac ( 22) Bigelow, C. C., and Geschwind, I. I., Compt. rend. tion, however, was inhibited irreversibly by tray. lab. Carlsberg, 31, 283 (1960) concentrations of urea more than 2 M. From (23) Weber, G., Biochem. J., 75, 345 (1960) measurements of ultraviolet difference spectra, (24) Konev, S. W., Doklady Acad. Nauk. U.S. S. R., 116, 594 (1957) fluorescence spectra and viscosity, the unfold (25) UImer, D. D., and Vallee, B. L., J. Biol. Chem, ing of the enzyme molecule was suggested to 236, 730 (1961) occur under these conditions. A good agree (26) Kagi, J. H. R., and Vallee, B. L., J. Biol. Chem., ment was found between the change in protein 235, 3188 (1960) structure and the irreversible loss of activity. (27) Hersh, R. T., Biochem. et Biophys. Acta, 58, 353 (1962) REFERENCES (28) Gilbert, G. A., Disc. Faraday Soc., 20, 68 (1955) (1) Swensen, A.D., and Boyer, P.D., J. Am. Chem. (29) Vallee, B. L., and Hoch, F. L., Proc. Natl. Acad. Soc., 79, 2174 (1957) Sci. U.S., 41, 327 (1955) Effects of Urea on Yeast Alcohol Dehydrogenase 89

(30) Wallenfels, K., Sund, H., Faessler, A., and 436 (1937) Burchard, W., Biochern. Z., 329, 31 (1957) (33) Nygaard, A. P., and Theorell, H., Acta. Chem. (31) Morales, M. F., J. Am. Chem. Soc., 77, 4169 Scand., 9, 1300 (1955) (1955) (34) Johnston, J.P., and Ogston, A.G., Trans. Faraday (32) Negelein, E., and Wulff, H.J., Biochem. Z., 289, Soc., 42, 789 (1946)