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Effect of crystallization upon the reactivity of horse liver alcohol dehydrogenase
J. Mol . Biol. (1966) 17, 513-524
Effect of Crystallization upon the Reactivity of Horse Liver Alcohol Dehydrogenaset H.
THEORELL
Nobel Medical Institute, Karolinska Institute, Stockholm, Sweden
B.
CHANCE AND
T.
YONETANI
Johnson Research Foundation, University of Pennsylvania Philadelphia, Penn., U.s.A. (Received 6 January 1966) The reactivity of suspensions of cryst a lline horse liver alc ohol dehydrogenase in the formation of its compounds with DPNH and with DPNH plus isobutyramide has been eva luated. Both spe ct rophot omet ric and fluorometric observations of the reaction s with DPNH show half-times between t en and twenty seconds at 3·5 sr-ammonium sulfate, whereas amorphous material under the same condit ions , or a solution of the enzyme in 2·5 M-amrnonium sulfate, gives much more rapid kinetics, in the latter case , lOW-fold more rapid. This change of reactivity closely follows t he solubility curve for t he crystalline material. Cal. culations of the diminution of reactivity expected from combined diffusion and chemical reaction in t he plate-like cryst als fail by a wide margin to explain the observed phenomenon. A similar low reactivity is observed in t he reaction of the enzyme-pyra zole compo und with DPN and 3·5 M·ammonium sulfate, but only a small increase of activity is observed at 2·5 M-ammonium sulfate. It is concluded that the crystalline st at e of the enzyme is much less reactive than the soluble material.
1. Introduction The striking success of X-ray crystallographers in determining the structure of some simple proteins at high resolutions, for example, ferrimyoglobin (Kendrew et al., 1960) and lysozyme (Blake, etal. 1965), already achieving a 5 A resolution on a structure of a protein as complicated as ferrihemoglobin (perutz, 1965), suggests that many enzymes will soon be studied in this fashion, for example, horse liver alcohol dehydrogenase (Branden, 1965). An important st art has been made on the st ruct ure of ligands bound to the heme, in the case of azide to the ferric form of the hemoprotein (Stryer, Kendrew & Watson, 1964), and in the case of ethyl isocyanide to the ferrous form of the hemoprotein (Nobbs , 1965). Th e results obtained so far suggest further use of this high-resolution t echnique in the determination of the structure of compounds resembling closely those of enzymes and subst rates (Keilin & Mann, 1936; Theorell, 1942; Chance, 1952), and in enzyme--eoenzyme compounds (Theorell & Bonnichsen, 1951; Theorell & Chanc e, 1951), thus permitting a further and more penetrating analysis of the mechani sm of enzyme action.
t
Wi th the t echnical assist a nce of T. Yosh ioka and A. RaviIIy.
513
514
H. THEORELL, B. CHANCE AND T. YONETANI
The detailed knowledge of structures affords opportunities for a deeper interpretation of protein and enzyme reactivity. A preliminary investigation of the reactivity of a suspension of crystals has been carried out with horse ferrimyoglobin and azide (Chance, Ravilly & Rumen, 1966), leading to the conclusion that crystallization has altered either the detailed configuration of the active site or the larger scale conformation of the protein to the extent that the reactivity of ferrimyoglobin toward azide has been reduced 20-fold. In order to determine whether this is an isolated phenomenon characteristic only of crystalline ferrimyoglobin or one pertaining generally to crystalline proteins, we present here a preliminary investigation of the reaction of suspensions of crystals of E with Rand O].
2. Experimental Methods (a) Spectrophotometry The techniques developed for the study of reaction kinetics of suspensions of whole cells and subcellular fragments seem to be ideally suited to studies of a suspension of crystals, and we have used the double-beam spectrophotometer, as it is relatively insensitive to the light-scattering properties of crystal suspensions (Chance, 1951). In addition, the double-beam spectrophotometer allows the observation of the kinetics of formation of the enzyme-coenzyme compound, without interference due to consecutive additions of the coenzyme. In the previous study, wavelengths of 328 and 353 to 354 mp' were found to give no deflection on adding R, due to the equal absorption of the R solution at these wavelengths. In these experiments, wavelengths of 360 and 321 to 325 tiu» were used in order to obtain equal response to the addition of R. A comparison of these pairs of wavelengths with the absorption spectrum of R suggests that both in previous experiments and those described in these papers, the absorbancy at the shorter wavelength is between 5 and 10% less than that expected from the absorption spectrum of a solution of R. This is attributed to stray or scattered light at the shorter wavelengths (321 to 328 m,..), in spite of the fact that a Wratten 39 stray-light filter was employed with the tungsten lamp. This small error makes no difference in the interpretation of the results, since the wavelengths were empirically selected on the basis that an addition of R caused no deflection of the output of the double-beam spectrophotometer, as is amply attested in the experimental traces of Fig. 2(a) and (b). The value of the molecular extinction coefficient for ER is not greatly affected by this small error, since the majority of the contribution occurs at 360 m,... The extinction coefficient for 325 to 360 mp' is computed from the data of Theorell & Yonetani (1963) to be 2·9 cm ! mN- i • (b) Fluorometry The employment of fluorometers for measuring the enhanced emission of R bound to the enzyme has been suggested previously (Boyer & Theorell, 1956; Theorell, 1962). In these studies we have employed a compensated fluorometer in which portions of the excitation light (366 mu) fall upon a fluorescence standard (Chance & Legallais, unpublished developments). The fluorescence of the enzyme-coenzyme compound is then compared with that of the fluorescence standard. An electronic circuit subtracts the output of the two photomultipliers, one measuring the standard and the other the R fluorescence, The fluorescence emission is measured at 407 ± 2 mp' in order to respond sensitively to EIR and insensitively to R. r
(c) Spectrophotometry in the ultraviolet region In order to measure the 0 compound in the presence ofpyrazole (Theorell & Yonetani, 1963) a double-beam spectrophotometer equipped with appropriate light source (deuterium
t Abbreviations used: ADH, liver alcohol dehydrogenase; E, enzyme (ADH); ER, ADHDPNH compound; I (IBA), isobutyramide; P, pyrazole; [ADH] in normal (N), co-enzyme binding equivalents/liter; 0, DPN; R, DPNH; EIR, enzyme isobutyramide-DPNH compound; EPO, enzyme pyrazole-DPN compound; ,.., ionic strength.
HORSE LIVER ALCOHOL DEHYDROGENASE
515
arc) was employed, so that satisfactory operation could be obtained at 325 and 295 txus: The molecular extinction coefficient for the EPO compound at 295 m,.. is 8 cm ! fiN-l. and the contribution at 325 mu is sufficiently small for this value to be employed without correction. r
(d) Calculations of the enzyme concentration
Fluorometric titrations of EI with R and spectrophotometric titrations of EP with 0 are used to determine the enzyme concentration. The values are expressed as normality. which is divided by 2 to obtain molarity.
3. Preparations ADH was purified on a carboxymethylcellulose column and found to be electrophoretically homogeneous and 90 to 95% pure, the remainder being due to "minor components". E was crystallized in 10% ethanol and the solutions were buffered with phosphate at pH 7'0 and 0·1 ionic strength at 4°e. The enzyme was assayed according to the method of Dalziel (1961). (Thanks are due to A. !keson and S. Taniguchi for their preparation of ADH and the solubility determinations.) (a) Control of crystal size
The crystals obtained in ethanol with phosphate buffer were centrifuged and resuspended in 3·5 M-neutralized ammonium sulfate at ooe. This procedure caused a fracturing of the crystals, and the size range extended from those not clearly visible in the microscope to thin needles or plates. The fractured crystals were centrifuged at low speed and the supernatant suspension consisted of thin plates and needles. having maximum dimensions in the range 5 to 15 JL and thicknesses of I to 2 JL or less, most of the crystals being too thin for accurate measurement «1 iL)' The crystals contain about 50% enzyme by volume, corresponding to a concentration of approximately 8 mx, of the same order as hemoglobin crystals. With this low concentration of the enzyme in the crystals, the possibility of self-absorption, which is a problem in the study of suspensions of ferrimyoglobin crystals, is not nearly so serious, and absorbancy of only 0·005 iL-1 at 325 miL is to be expected for the ER compound. Since most of the crystals were no greater than this along their shortest dimension, self-absorption is unlikely to be a source of error in studies of this material. (b) Solubility determinations
In order to determine the exact values of ammonium sulfate concentration at which the soluble or crystalline protein could be maintained, the "salting out" curves for E, ER and ERI were determined as follows: 0·1 ml, of the stock solution of the enzyme, electrophoretically homogeneous and at a concentration of 220 JLN, was added to 0-4 ml. of cold ammonium sulfate solution slowly and with gentle stirring to give a final concentration of ammonium sulfate in the range of 1·2 to 3·2 M. The tubes were equilibrated at room temperature of 20 0 e for 45 minutes and then centrifuged at 4000 rev.jmin for 15 minutes to obtain a clear supernatant fraction. The activity remaining in the supernatant fraction was assayed by the procedure of Dalziel (1961). Similar experiments were carried out with the ER complex formed by the addition of excess R to the above-mentioned stock solution of E (5 p.l. of 20 mM-R was added to 100 iLl. of E, giving a final concentration of R of 1 mx), The same procedure was followed and the supernatant fraction was assayed as before. An examination of the precipitate in both cases indicated it to be amorphous
516
H. THEORELL, B. CHANCE AND T. YONETANI
under the microscope, but the suspensions showed birefringence when stirred. In a third series of experiments, the EIR intermediate was formed in the presence of 60 mM-I and 500 fLM-R. The experimental results are indicated in Fig. 1, where the values of the ordinates are in molarity of ammonium sulfate and expressed in mg. of E/ml. of supernatant fluid. The graph illustrates a typically doubly broken characteristic of a salting out curve. The first break for E occurs at 2'4 M-ammonium sulfate and at 2·6 Mfor ER. The second break is observed at 2·9 Mfor E and at 3·1 Mfor ER. The salting out curve for EIR lies between these two curves. The curves of Fig. 1 apply with a reasonable
2'0 oJ
c:: 0 0
....
.,E
Q.
~ '-
0
.:§ 1'0 :r: 0 <:
-' OJ
E
0
4
FIG. 1. Solubility curves for E, ER, and EIR; the concentration at E indicated by the values of the ordinates; the concentration of R is 500 I'M and of I (isobutyramide) 60 mx, The temperature is 23·5°C; the buffer is 0·1 I' phosphate (pH 7,0). By courtesy of A. Akeson and S. Taniguchi.
degree of accuracy to the conditions of our experiments, particularly those of Fig. 5 where at 2·9 M-ammonium sulfate concentration, two-thirds of the crystals of E dissolved at 25°C. This solubility should be expected at 2·5 M-ammonium sulfate, as in Fig. 1. However, the 5°C higher temperature and the fact that the crystalline and not the amorphous material was used in Fig. 5 can account for the difference in the molarity of ammonium sulfate. The solubility increase due to the formation of ER is surely to be taken into account in the range between 2·5 and 3·1 M-ammo.nium sulfate and leads to additional kinetic complexity. It is apparent from both Figs 1 and 5 that the use of 3·5 M-ammonium sulfate effectively prevented solubility changes in a particular experiment as being a factor in the reaction kinetics. (c) Experimental design
The basic experimental design involves a comparison of the kinetics of a compound formation of the crystal suspension with the kinetics of the soluble material under identical conditions. While this has been possible with ferrimyoglobin because of the slow solubility of the crystals (Chance et al., 1966), the more rapid solubility of the E crystals prevents this approach. Instead we have studied the kinetics of a heterogeneous system in which varying portions of E were in solution or in the crystalline or amorphous form. For example, if the protein obtained as described above is diluted in 2·9 M-ammonium sulfate, about half is in solution and half in
HORSE LIVER ALCOLHOL DEHYDROGENASE
517
the precipitated state at 20°C (Fig. I). If on the other hand, a solution of E is added to 3 M-ammonium sulfate, a portion will be in solution and the remainder either as an amorphous or microcrystalline precipitate; the precipitates obtained in the experiments of Fig. 1 had a crystalline sheen although no crystals could be resolved microscopically.
4. Results (a) Spectroscopic studies Figure 2 illustrates the effect upon the formation of ER of ammonium sulfate concentrations below and above the solubility values for the crystals. In this experiment, 2·4 fLN-crystalline E was added to 2'5, 3·0 and 3·5 M-ammonium sulfate and treated with two equivalents of R (4,8 fLM). From Fig. 2(a}, at 2·5 M-ammonium
J..
325 to 360 mJ1'
~Inereased absorption at 325
17 sec+!
1+
17 see-l
log Joll =0,005 (2 em path)
t
mf'
I-
17 see-J
~
I
I-
\
2'4 fLN-E '-
...
'n.
o.
UJ
~.
t
.
..
I
.di
j
.-
4,.8 j.£M-DPNH
4·8 fLM-DPNH
2·5 M-Ammonium sulfate
3·0 M-Ammonium sulfate
(0)
(b)
..
i
4,8 fLM-DPNH
3·5 M-Ammonium sulfate (c)
FIG. 2. Kinetics and extent of formation of ER at various concentrations of ammonium sulfate. The concentrations of E and R are indicated in the diagram. The ammonium sulfate was buffered with 0·1 Il- phosphate buffer (pH 7,0). A downward deflection of the traces indicates a decrease of absorbanoy at 360 mil- with respect to 325 mil-. The sequential additions of DPNH in (a) and (b) indicate that the absorbancy of DPNH itself is balanced out at the wavelength pair selected. The bottom trace shows time marks every 5 sec. Experiments in an open cuvette of a 2-cm optical path; volume 6 ml.; temperature 25°C. (1216.12, 10, 6 IV.)
sulfate the enzyme is in solution and the addition of two equivalents of R caused an abrupt drop of the trace within the mixing time, while subsequent additions caused no further reaction. The absorbancy calibration indicates the formation of 2·5 fLN-ER. In 3 M-ammonium sulfate (Fig. 2(b)} the addition of two equivalents of R caused a rapid drop, as in the previous experiment, except that the amplitude of the rapid phase was only two thirds of the maximal excursion of the trace; the remaining third of the reaction proceeds in a slower phase with a half-time of about ten seconds. The total amount of ER formed under these conditions is 2·5 fLN. Only 1·6 fLN is formed in the rapid step, just a little over half the total enzyme. In 3·5 M-ammonium sulfate (Fig. 2(c)}, there is no rapid phase of absorbancy change caused by the addition of R, only the slow phase was recorded. Furthermore, the half-time of the slow phase is approximately eleven seconds, comparable to that
518
H. THEORELL, B. CHANCE AND T. YONETANI
of the slow phase in 3 M-ammonium sulfate. The total absorbancy change observed in the reaction is diminished to approximately half the value obtained in the preceding two charts. We have already considered the possibility that the absorption of ER in the crystalline material may be less than that in the solution, but believe that this is unlikely because ofthe thinness of the crystals and the relatively small concentrations of E that they contain (8 ma). Furthermore, the fluorometric data of Fig. 5 suggest that there is no decrease in the amount of ER formation; the amplitude of the fluorescence change is undiminished in the crystalline material. We have therefore, at present, no explanation for the diminished extent of the reaction of Fig. 2(c). However, since our attention here is concentrated upon the kinetic aspects, we are able to compare the rate of reaction of Fig. 2(c) with the rate that would have been observed with the rate of Fig. 2(a) (when measurements with the rapid flow apparatus are then taken into account (Fig. 4)). In summary, a progressive increase of ammonium sulfate concentration results in obliteration of the rapid phase of the reaction and its replacement by a reaction with a half- time of ten seconds. A comparison of the development of the slow phase of the reaction with the salting out curve for the enzyme (see Fig. 1) clearly indicates a relationship between the state of crystallization of the enzyme and its reduced rate of reaction with the coenzyme. If, instead of the crystalline material, amorphous E is added to 3·5 M·ammonium sulfate, the results seen in Fig. 3(a) and (b) are obtained. At 3 M-ammonium sulfate, the addition of 4'8 /kM-R to 1·7 /kN-E caused rapid formation of ER, and addition of a further equivalent of R caused no further reaction. In 3·5 M.ammonium sulfate, addition of 4·8 /kM-R to 2·6 /kN.E caused a rapid absorbancy change corresponding to 1·7 /kN.ER; however, this was followed by a considerably slower reaction. Here we may tentatively attribute the rapid portion of the reaction to the amorphous
I
325 to 360 mJL ,Increased absorption at 325 m/k
1T
log Jo/ l = 0·005 (2 em optical path)
~17 sec
1'4-17 sec
r-l
2'6/-1-N-E
r+
1'7f!'N-E
r
-..j
IV'-
~
~~,
.....
-
...., "I.
U
4'8/kM-DPNH
,
-
....--
--
i\"I "lr1W11~
4·8JLM-DPNH
3·0 M-Ammonium sulfate
3·5 M-Ammonium sulfate
(a)
(b)
FIG. 3. illustrating the effect of ammonium sulfate concentration upon the reactivity of amorphous ADH. Other conditions identical to those of Fig. 2. (1216-9, 8 IV.)
HORSE LIVER ALCOHOL DEHYDROGENASE
519
precipitate of the protein. The slower portion ofthe trace may be due to the formation of a microcrystalline precipitate of extremely low reactivity (see p. 524). (b) Rate of formation of E R
Figure 4 illustrates the high reactivity of E towards R in 2·5 M-ammonium sulfate. This trace has been obtained with the regenerative flow apparatus (Chance, 1964) and the main chamber is filled with ,....,.,1 fLN-horse liver E. The small syringe is filled
1+sao msec RY~4msec --.j
Flow ... velocity
I--
1-
i\
1-
/-- ' -
......... -.'
log 10/1 = 0'002
-
T
t
10fLM-DPNH
FIG. 4. Illustrating the rapid combination of E and R in 2·5 xr-ammonium sulfate. Approximately lIJoN.E mixed with 800 IJoM·R, with 360 IJoM·forlllaldehyde present. The top trace indicates increasing flow velocity of a downward deflection. The maximum deflection corresponds to a time after mixing of 4 msec. The wavelengths are 321 and 360 mu.; temperature 25°C; 0·1 IJo phosphate buffer. (1211·4 IV.)
with 800 fLM-R and since the dilution factor is 80-fold, 10 p.M-R was delivered into the observation tube. Upon initiating the flow, the flow velocity indicator (top trace) indicated a maximal deflection corresponding to a time after mixing of approximately four milliseconds. The spectrophotometric trace (somewhat delayed with respect to the flow velocity trace because ofthe long averaging time employed in order to record the small optical density change (O'002jdivision)) recorded a downward deflection to a decreased level of absorption at 360 mfL and remained roughly constant until the flow stopped, when the larger increment of absorption was observed. The total change corresponded to the formation of 1 fLN-ER. The enzyme intermediate remains in a steady state of turnover activated by the presence of a small (360 fLM) concentration of formaldehyde, until R is exhausted and free E is formed. It is for this reason that the observation chamber was initially filled with free E, and not ER resulting from the previous experiments. The data of this and other experiments indicate approximately half completion of the reaction at four milliseconds. This value may be compared to the half-time of ten seconds for the slow phase of formation of ER in 3 and 3·5 M-ammonium sulfate with 4·8 fLM-R. At equal concentrations of R, the ratio of half-times is about 1000-fold. The velocity constant for the formation of ER calculated from Fig. 4 is about 2 X 107 M -1 sec - \ in better agreement with the value of 1·1 X 107 measured from the over-all reaction (Theorell & McKinley-McKee, 1961) than with that determined in our earlier experiments (Theorell & Chance, 1951). However, the main purpose of this demonstration is to show the high reactivity of the soluble form of E at high ammonium sulfate concentrations similar to those employed in the preceding Figure.
520
H. THEORELL, B. CHANCE AND T. YONETANI
(C) Fluoromeiric studies
Since it is possible that the nature of the ER compounds formed at high concentrations of ammonium sulfate would differ from those formed at lower ionic strength, and also since it is of interest to determine that the R is bound to the crystalline enzyme in such a way as to enhance its fluorescence at the high ammonium sulfate concentrations, we have made some studies of the kinetics of the fluorescence changes under the conditions of Fig. 2. Figure 5(a) to (d) represents a series of titrations of E with R in the presence of 2·9 mx-I, In the absence of ammonium sulfate and the presence of 0·1 M-phosphate, sequential additions of 0·8 JLM-R gave an end point to the titration very close to 1·2 JLN-E.
I
I
-
~'-= '---.
2·9 mM-IBA
I
1-1 min
I
I
I I
2'9 M-Ammonium
su lfate (c)
FIG. 5. Fluorometric measurements of the effect of ammonium sulfate concentration upon the combination of EI and R in the presence of 2·9 mM-I. The concentrations of E and R added are indicated in the Figure 0·1 ,.,. phosphate buffer; 25°C. (1213-13,19,21,24 IV.)
When crystals were added to 0·1 JLM-phosphate buffer, they dissolved immediately; addition of 1·2 JLN-E caused no significant deflection of the trace, nor was there any deflection when 2·9 mM-I was added. The first addition of 0·8 fLM.R gave two thirds of the maximal deflection, and a plateau was reached after three more additions of the coenzyme. The fluorescence increase per 0·8 fLM-E is 3·3 scale divisions. The reaction of R is so rapid that it occurs within the mixing time. In the experiment of Fig. 5(b), 1·5 fLN-crystalline enzyme was added to 2·5 M-ammonium sulfate. The upward deflection of the trace is attributed to the dissolving of the crystals; the crystals caused a considerable scattering of the excitation into the measuring photomultiplier, but as they dissolved, the scattering diminished with a half-time of about ten seconds. The total change of fluorescence due to solution of the enzyme was 2·0 large scale divisions. Addition of I caused no further change, and titration with 0·8 fLM-R caused a rapid increase of fluorescence of 3·3 scale divisions as in Fig. 5(a). The reactions were again so fast that the rate was limited by the mixing time. In the experiment of Fig. 5(c), 2·9 M-ammonium sulfate was employed. The initial addition of E gave a deflection of 2·7 scale divisions. There followed a much slower return of the trace than in Fig. 5(b) and the extent of the return was 1·7 scale divisions.
HORSE LIVER ALCOHOL DEHYDROGENASE
521
At the time of the I and R additions, a portion of the material equivalent to one scale division can be considered to have remained in the crystalline state. The first addition of R gave a rapid deflection of 3·1 scale divisions. The three succeeding additions caused a further fluorescence increase of 2 scale divisions and showed less rapid kinetics than in the previous experiments. In Fig. 5(d) at 3·5 M-ammonium sulfate there was no decrease of deflection following the addition of E. The crystals remain as such under these conditions (see Fig. 1). Addition of 0·8 fLM-R caused a total deflection of 32 divisions, as in the other cases. Now, however, the rapid portion of this trace is almost imperceptible and the halftime ofthe reaction is about 20 seconds. Further additions ofR complete the titration, but in each case it is apparent that a slow change was involved. It is appropriate to compare the results of Fig. 5(d) with those of Fig. 2(c). The slower rate in Fig. 5(d) is due to the lower concentrations of both E and R employed in the fluorometric experiment. Thus, the reactivity of the crystalline material is very small whether measured spectrophotometrically or fluorometrically. (d) Reactivity towards isobutyramide
While the data of Figs 2 and 5 demonstrate the low activity of the crystalline enzyme towards R, the reaction ofER and I, a smaller molecule, is presented in Fi~. 6. In Fig. 6 we have employed the fluorometric technique of Fig. 5 and the ER compound is formed from 1·2 fLN-E and 2·4 fLM-R in 3·5 M-ammonium sulfate. The trace then shows the increase of fluorescence caused by the addition of 2·9 mM-I to ER. An increase of fluorescence (4 scale divisions) similar to the fluorescence increase of Fig. 5(d) is seen, where R is added to EI. The rate appears, however, to be more rapid; the first three divisions of deflection occur in the mixing time and only the last 20% occurs in a measurable slow reaction.
T
0·8 fLN-ERI
1 FIG. 6. Fluorometric determination of the rate of combination of 1·2 I"N-ER with 2·9 mM-I in 3·5 msr-ammonium sulfate. 0·11" phosphate buffer (pH 7'0); temperature 25°C. (1213-17 IV.)
It is difficult to compare this reaction with the data of Fig. 5. The ratio of I to R employed in the two experiments is approximately 3000:1 and thus a fairly rapid reaction would be expected in Fig. 6. While somewhat lower concentrations of I can be used, the larger dissociation constant for this substance prevents its use at concentrations of the same order as employed in the reaction of EI plus R. Figure 6 does demonstrate that even with as high a concentration of I as 2·9 mx, a portion of the reaction requires more than a minute for its completion.
H. THEORELL, B. CHANCE AND T. YONETANI
522
(c) Reaction of 0 with the enzyme-pyrazole compound
Figure 7 shows a series of traces at increasing ammonium sulfate concentrations, similar to those of Figs 2 and 5. Here the reaction of the enzyme-pyrazole compound (EP) with 0 is recorded in terms of the increased absorbancy change at 325 mfL, using 395 mfL as the reference wavelength (Theorell & Yonetani, 1963). In trace (a), 295 to 325 mfL Increased absorption at 295 mIL optical path 2 em
',4
fLJ1Mj-j~PN 2.8_ .-
r
.+
jiM_DPN
~ 2'8jtM-DPN
log 1011= 0·01
-+~
IC!'4~Eti T~,!!o$ log 101[= 0-\1
r- T -l ~~c I+L-
No ammonium sulfate (a)
=t=t==
-_-+j sec 60 I+-_ I I
2·5 M-Ammonium sulfate ( b)
t I fL
I
M -DPN
i'
.if--'·8 I~ I log [011-0'01 r---
~
60 _ -+j sec I+-_
I
I
3·0 M-Ammonium sulfate (c)
3·5 M-Ammonium sulfate (d)
FIG. 7. Spectrophotometric determinations of the reactivity of EP with 0; 1·4 mM.P; concentrations of E and 0 indicated in the Figure. A downward deflection indicates increased absorbtion at 295 mu relative to 329 tau, 0·1 ,.,. phosphate buffer (pH 7'0); temperature 25°C. (1216A, 3, 14, 9,10 IV.)
several portions of 1·4 fLM-O are added to 1·2 fLN-E. The abrupt downward deflection of the trace indicates the combination time in the absence of ammonium sulfate to be less than the mixing time. More than 80% of the maximal deflection is obtained with one equivalent of 0, and the total absorbancy change corresponds closely with the formation of 1·2 fLN-EPO. In trace (b) there is a single addition of 2·8 fLM-O to 2·4 fLN-enzyme in 2·5 M-ammonium sulfate. In this case, the rate of formation of the EPO compound is readily recorded, and the reaction occurs at a measurable rate with soluble enzyme in 2·5 M-ammonium sulfate. It is possible that sulfate itself has inhibited the reaction somewhat. At 3·0 M-ammonium sulfate (Fig. 7(c)) the reaction is somewhat slower and the extent is smaller. Finally, at 3·5 M.ammonium sulfate (Fig. 7(d)), where the enzyme is all in the crystalline form, the amplitude is further reduced and the rate further slowed. There is some evidence under these conditions that the reaction is biphasic, a feature not observed in the kinetics of Figs 2 and 5. In summary, we find the reaction of EP with 0 to be quite slow in 3·5 M-ammonium sulfate.
5. Discussion The striking effect of a crystallization upon the speed of combination of ADH with DPNH as measured spectrophotometrically or fluorometrically in the presence of I supports and greatly increases the generality of the conclusions based upon studies of a suspension of ferrimyoglobin crystals with azide (Chance et al., 1966). In spite of the fact that, in the case ofE, it was not possible to measure completely crystalline
HORSE LIVER ALCOHOL DEHYDROGENASE
523
and completely soluble materials at the same concentrations of ammonium sulfate, studies of a gradation of effects with increased ammonium sulfate follow the "salting out" curve for the enzyme so closely that the decrease of reactivity of E with increasing ammonium sulfate concentration can be attributed to the preservation of crystal form at the higher concentrations. The extent of decrease of reactivity of E can be quantitated for the reaction with R; the ratio of the half-times of the reaction of E with equal concentrations of R at 3·5 and 2·5 M-ammonium sulfate is in excess of 1000. The ratio of the half-times in the reaction with 0 in the presence of pyrazole at 3·5 and 2·5 M.ammonium sulfate is about tenfold. This is a surprising result, because EIR and EPO are crystallographically isomorphic, and one would have expected that the steric and other factors involved in their formation in the crystal state would have been identical. As a matter of fact, the half times of EIR and EPO formation are not greatly different at 3·5 M-ammonium sulfate. However, the fact that the EP 0 reaction proceeds at a measurable velocity at 2·5 M-ammonium sulfate suggests that another factor is operative in this case. A possible explanation is afforded by the existence of an induction period in the formation of the pyrazole complex which is absent in the case of the R reaction of E and I (Theorell & Yonetani, 1963). Many factors involved in the binding of the enzyme and coenzyme could be altered on crystallization, and thereby provide an explanation of the decreased reactivity of the crystalline material. Diffusion ofthe coenzyme through a water phase to the active site appears unlikely to be a significant factor in the decreased reactivity of the crystalline material. The existence of a water phase in the crystal is indicated by the observation of 48% water content in the unit cell. The diminution ofthe R concentration due to combined diffusion and reaction velocity in this water phase can be calculated for the thin plates of E by an equation which is considerably simplified compared to that used for the calculation of the cylindrical geometry of the ferrimyoglobin crystals (Roughton, 1932). (Thanks are due to Dr R. Foster for assistance in the diffusion calculations.) The ratio of the R concentration in the crystal and in the surrounding solution is given by the following expression, using the terminology of Appendix I (Chance et al., 1966): [R] mean inside 1 [R] at surface
+
!-JkE
2])
where t is the thickness of the crystal; k, the second-order velocity constant for the combination ofE and R; E, the enzyme concentration; and D, the diffusion coefficient of R. Since most of the crystals were of a thickness of 11-' or less, tj2 is 5 X 10 - 5 em, The second-order velocity constant is taken to be 1 Xl0 7 M- 1 sec- 1 (see above). The concentration of E in the crystals is approximately 8 mM. The diffusibility of R is decreased compared to azide by the square root of the ratio of the molecular weights (-\/17) or 2·5 X 10- 6 cm 2jsec. A substitution of these values into the equation indicates that the azide concentration within the plates is 0·12 that at its surface. It is apparent that the thinness of the plates and the low concentration of E in the crystals compensates for the high reaction velocity constant of combination and the low diffusibility of R. Consequently, the conditions for observing the reaction of E and R in crystals of E without interference from diffusion are only fourfold less
524
H. THEORELL, B. CHANCE AND T. YONETANI
favorable than those for the observation of the reaction of ferrimyoglobin and azide. It is of great interest that the combined reaction and diffusion equation fails much more seriously in this case to explain the slowness of the observed reaction of E and R; if, indeed, the equation afforded an explanation, we would at least expect a proportionality between the calculated ratios of concentrations (inside and outside) and the observed values of k. This is not the case; for azide, the ratio is 0·5 x21 =11, and for this case the ratio is 0·12 X1000=120. Thus, the "fit" is ten times worse for the reaction of E and R. Since we may conclude that k is decreased, the ratios of inside to outside oonoentrations must be even nearer unity, or the tolerance of error of the diffusion constant much greater. For example, for k = 3x104 M- 1 seo" ", D may fall to 2·5Xl0- 8 cm 2/sec to give the same inside-outside ratio calculated above. The possibility that the crystals are completely unreactive and that the reactivity observed is due to the rate of solution and recrystallization of the enzyme is to be considered. The experimental data suggest, however, that this is not the principal factor in these particular circumstances. In Fig. 2(b) and (0), where the ammonium sulfate concentration is increased from 3·0 to 3,5, the half-time for the slow phase is not greatly decreased. However, Fig. I shows that these two sulfate concentrations would very greatly decrease the amount of free enzyme in equilibrium with the crystals. The possibility that the crystals may have become coated with a monolayer of protein impurity has also to be considered. The slow portion of the kinetics of Fig. 3 is helpful on this point, and suggests that a microcrystalline material of a very large surface-to-volume ratio became "coated" to the same extent as the larger crystals of Fig. l(c), which we regard as very unlikely. REFERENCES Blake, C. C. F., Koenig, D. F., Mair, G. A., North, A. C. T., Phillips, D. C. & Sanna, V. R. (1965). Natvre, 206,757. Boyer, P. & Theorell, H. (1956). Acta Ohem, Scand. 10, 447. Branden, C. 1. (1965). Archiv. Biochem, Biophys. 112, 215. Chance, B. (1951). Rev. Sci. Lnstr., 22, 619. Chance, B. (1952). In Modern Trends in Physiology and Biochemistry, ed. by E. S. G. Baron, p. 25. New York: Academic Press. Chance, B. (1964). In IUB/IUBS Rapid Mixing and Sampling Techniques in Biochemistry, ed. by B. Chance, Q. H. Gibson, R. Eisenhardt & K. K. Lonberg-Holm, p. 125. New York: Academic Press. Chance, B., Ravilly, A. & Rumen, N. (1966). J. Mol. Biol. 17, 525. Dalziel, K. (1961). Biochem, J. 80, 440. Keilin, D. & Mann, T. (1936). Proc, Roy. Soc. B, 122, 119. Kendrew, J. C., Dickerson, R. E., Strandberg, B. E., Hart, R. G., Davies, D. R., Phillips, D. C. & Shore, V. C. (1960). Nature, 185, 422. Nobbs, C. L. (1965). J. Mol. Biol. 13, 325. Perutz, M. F. (1965). J. Mol. Biol. 13, 646. Roughton, F. J. W. (1932). Proc. Roy. Soc. B, Ill, 1. Stryer, L., Kendrew, J. C. & Watson, H. C. (1964). J. Mol. Biol. 8, 96. Theorell, H. (1942). Enzymologia Acta Biocatalytica, 10, 250. Theorell, H. (1962). Pontijicae Academiae Scientiarum Scripta Varia, 22, 1. Theorell, H. & Bonnichsen, R. (1951). Acta Ohem, Scand. 5, 1l05. Theorell, H. & Chance, B. (1951). Acta Ohern; Scand. 5, 1127. Theorell, H. & McKinley-McKee. (1961). Acta, Ohem. Scand. 151797. Theorell, H. & Yonetani, T. (1963). Biochem, Zeit. 338, 537.
Effect of Crystallization upon the Reactivity of Horse Liver Alcohol Dehydrogenaset H.
THEORELL
Nobel Medical Institute, Karolinska Institute, Stockholm, Sweden
B.
CHANCE AND
T.
YONETANI
Johnson Research Foundation, University of Pennsylvania Philadelphia, Penn., U.s.A. (Received 6 January 1966) The reactivity of suspensions of cryst a lline horse liver alc ohol dehydrogenase in the formation of its compounds with DPNH and with DPNH plus isobutyramide has been eva luated. Both spe ct rophot omet ric and fluorometric observations of the reaction s with DPNH show half-times between t en and twenty seconds at 3·5 sr-ammonium sulfate, whereas amorphous material under the same condit ions , or a solution of the enzyme in 2·5 M-amrnonium sulfate, gives much more rapid kinetics, in the latter case , lOW-fold more rapid. This change of reactivity closely follows t he solubility curve for t he crystalline material. Cal. culations of the diminution of reactivity expected from combined diffusion and chemical reaction in t he plate-like cryst als fail by a wide margin to explain the observed phenomenon. A similar low reactivity is observed in t he reaction of the enzyme-pyra zole compo und with DPN and 3·5 M·ammonium sulfate, but only a small increase of activity is observed at 2·5 M-ammonium sulfate. It is concluded that the crystalline st at e of the enzyme is much less reactive than the soluble material.
1. Introduction The striking success of X-ray crystallographers in determining the structure of some simple proteins at high resolutions, for example, ferrimyoglobin (Kendrew et al., 1960) and lysozyme (Blake, etal. 1965), already achieving a 5 A resolution on a structure of a protein as complicated as ferrihemoglobin (perutz, 1965), suggests that many enzymes will soon be studied in this fashion, for example, horse liver alcohol dehydrogenase (Branden, 1965). An important st art has been made on the st ruct ure of ligands bound to the heme, in the case of azide to the ferric form of the hemoprotein (Stryer, Kendrew & Watson, 1964), and in the case of ethyl isocyanide to the ferrous form of the hemoprotein (Nobbs , 1965). Th e results obtained so far suggest further use of this high-resolution t echnique in the determination of the structure of compounds resembling closely those of enzymes and subst rates (Keilin & Mann, 1936; Theorell, 1942; Chance, 1952), and in enzyme--eoenzyme compounds (Theorell & Bonnichsen, 1951; Theorell & Chanc e, 1951), thus permitting a further and more penetrating analysis of the mechani sm of enzyme action.
t
Wi th the t echnical assist a nce of T. Yosh ioka and A. RaviIIy.
513
514
H. THEORELL, B. CHANCE AND T. YONETANI
The detailed knowledge of structures affords opportunities for a deeper interpretation of protein and enzyme reactivity. A preliminary investigation of the reactivity of a suspension of crystals has been carried out with horse ferrimyoglobin and azide (Chance, Ravilly & Rumen, 1966), leading to the conclusion that crystallization has altered either the detailed configuration of the active site or the larger scale conformation of the protein to the extent that the reactivity of ferrimyoglobin toward azide has been reduced 20-fold. In order to determine whether this is an isolated phenomenon characteristic only of crystalline ferrimyoglobin or one pertaining generally to crystalline proteins, we present here a preliminary investigation of the reaction of suspensions of crystals of E with Rand O].
2. Experimental Methods (a) Spectrophotometry The techniques developed for the study of reaction kinetics of suspensions of whole cells and subcellular fragments seem to be ideally suited to studies of a suspension of crystals, and we have used the double-beam spectrophotometer, as it is relatively insensitive to the light-scattering properties of crystal suspensions (Chance, 1951). In addition, the double-beam spectrophotometer allows the observation of the kinetics of formation of the enzyme-coenzyme compound, without interference due to consecutive additions of the coenzyme. In the previous study, wavelengths of 328 and 353 to 354 mp' were found to give no deflection on adding R, due to the equal absorption of the R solution at these wavelengths. In these experiments, wavelengths of 360 and 321 to 325 tiu» were used in order to obtain equal response to the addition of R. A comparison of these pairs of wavelengths with the absorption spectrum of R suggests that both in previous experiments and those described in these papers, the absorbancy at the shorter wavelength is between 5 and 10% less than that expected from the absorption spectrum of a solution of R. This is attributed to stray or scattered light at the shorter wavelengths (321 to 328 m,..), in spite of the fact that a Wratten 39 stray-light filter was employed with the tungsten lamp. This small error makes no difference in the interpretation of the results, since the wavelengths were empirically selected on the basis that an addition of R caused no deflection of the output of the double-beam spectrophotometer, as is amply attested in the experimental traces of Fig. 2(a) and (b). The value of the molecular extinction coefficient for ER is not greatly affected by this small error, since the majority of the contribution occurs at 360 m,... The extinction coefficient for 325 to 360 mp' is computed from the data of Theorell & Yonetani (1963) to be 2·9 cm ! mN- i • (b) Fluorometry The employment of fluorometers for measuring the enhanced emission of R bound to the enzyme has been suggested previously (Boyer & Theorell, 1956; Theorell, 1962). In these studies we have employed a compensated fluorometer in which portions of the excitation light (366 mu) fall upon a fluorescence standard (Chance & Legallais, unpublished developments). The fluorescence of the enzyme-coenzyme compound is then compared with that of the fluorescence standard. An electronic circuit subtracts the output of the two photomultipliers, one measuring the standard and the other the R fluorescence, The fluorescence emission is measured at 407 ± 2 mp' in order to respond sensitively to EIR and insensitively to R. r
(c) Spectrophotometry in the ultraviolet region In order to measure the 0 compound in the presence ofpyrazole (Theorell & Yonetani, 1963) a double-beam spectrophotometer equipped with appropriate light source (deuterium
t Abbreviations used: ADH, liver alcohol dehydrogenase; E, enzyme (ADH); ER, ADHDPNH compound; I (IBA), isobutyramide; P, pyrazole; [ADH] in normal (N), co-enzyme binding equivalents/liter; 0, DPN; R, DPNH; EIR, enzyme isobutyramide-DPNH compound; EPO, enzyme pyrazole-DPN compound; ,.., ionic strength.
HORSE LIVER ALCOHOL DEHYDROGENASE
515
arc) was employed, so that satisfactory operation could be obtained at 325 and 295 txus: The molecular extinction coefficient for the EPO compound at 295 m,.. is 8 cm ! fiN-l. and the contribution at 325 mu is sufficiently small for this value to be employed without correction. r
(d) Calculations of the enzyme concentration
Fluorometric titrations of EI with R and spectrophotometric titrations of EP with 0 are used to determine the enzyme concentration. The values are expressed as normality. which is divided by 2 to obtain molarity.
3. Preparations ADH was purified on a carboxymethylcellulose column and found to be electrophoretically homogeneous and 90 to 95% pure, the remainder being due to "minor components". E was crystallized in 10% ethanol and the solutions were buffered with phosphate at pH 7'0 and 0·1 ionic strength at 4°e. The enzyme was assayed according to the method of Dalziel (1961). (Thanks are due to A. !keson and S. Taniguchi for their preparation of ADH and the solubility determinations.) (a) Control of crystal size
The crystals obtained in ethanol with phosphate buffer were centrifuged and resuspended in 3·5 M-neutralized ammonium sulfate at ooe. This procedure caused a fracturing of the crystals, and the size range extended from those not clearly visible in the microscope to thin needles or plates. The fractured crystals were centrifuged at low speed and the supernatant suspension consisted of thin plates and needles. having maximum dimensions in the range 5 to 15 JL and thicknesses of I to 2 JL or less, most of the crystals being too thin for accurate measurement «1 iL)' The crystals contain about 50% enzyme by volume, corresponding to a concentration of approximately 8 mx, of the same order as hemoglobin crystals. With this low concentration of the enzyme in the crystals, the possibility of self-absorption, which is a problem in the study of suspensions of ferrimyoglobin crystals, is not nearly so serious, and absorbancy of only 0·005 iL-1 at 325 miL is to be expected for the ER compound. Since most of the crystals were no greater than this along their shortest dimension, self-absorption is unlikely to be a source of error in studies of this material. (b) Solubility determinations
In order to determine the exact values of ammonium sulfate concentration at which the soluble or crystalline protein could be maintained, the "salting out" curves for E, ER and ERI were determined as follows: 0·1 ml, of the stock solution of the enzyme, electrophoretically homogeneous and at a concentration of 220 JLN, was added to 0-4 ml. of cold ammonium sulfate solution slowly and with gentle stirring to give a final concentration of ammonium sulfate in the range of 1·2 to 3·2 M. The tubes were equilibrated at room temperature of 20 0 e for 45 minutes and then centrifuged at 4000 rev.jmin for 15 minutes to obtain a clear supernatant fraction. The activity remaining in the supernatant fraction was assayed by the procedure of Dalziel (1961). Similar experiments were carried out with the ER complex formed by the addition of excess R to the above-mentioned stock solution of E (5 p.l. of 20 mM-R was added to 100 iLl. of E, giving a final concentration of R of 1 mx), The same procedure was followed and the supernatant fraction was assayed as before. An examination of the precipitate in both cases indicated it to be amorphous
516
H. THEORELL, B. CHANCE AND T. YONETANI
under the microscope, but the suspensions showed birefringence when stirred. In a third series of experiments, the EIR intermediate was formed in the presence of 60 mM-I and 500 fLM-R. The experimental results are indicated in Fig. 1, where the values of the ordinates are in molarity of ammonium sulfate and expressed in mg. of E/ml. of supernatant fluid. The graph illustrates a typically doubly broken characteristic of a salting out curve. The first break for E occurs at 2'4 M-ammonium sulfate and at 2·6 Mfor ER. The second break is observed at 2·9 Mfor E and at 3·1 Mfor ER. The salting out curve for EIR lies between these two curves. The curves of Fig. 1 apply with a reasonable
2'0 oJ
c:: 0 0
....
.,E
Q.
~ '-
0
.:§ 1'0 :r: 0 <:
-' OJ
E
0
4
FIG. 1. Solubility curves for E, ER, and EIR; the concentration at E indicated by the values of the ordinates; the concentration of R is 500 I'M and of I (isobutyramide) 60 mx, The temperature is 23·5°C; the buffer is 0·1 I' phosphate (pH 7,0). By courtesy of A. Akeson and S. Taniguchi.
degree of accuracy to the conditions of our experiments, particularly those of Fig. 5 where at 2·9 M-ammonium sulfate concentration, two-thirds of the crystals of E dissolved at 25°C. This solubility should be expected at 2·5 M-ammonium sulfate, as in Fig. 1. However, the 5°C higher temperature and the fact that the crystalline and not the amorphous material was used in Fig. 5 can account for the difference in the molarity of ammonium sulfate. The solubility increase due to the formation of ER is surely to be taken into account in the range between 2·5 and 3·1 M-ammo.nium sulfate and leads to additional kinetic complexity. It is apparent from both Figs 1 and 5 that the use of 3·5 M-ammonium sulfate effectively prevented solubility changes in a particular experiment as being a factor in the reaction kinetics. (c) Experimental design
The basic experimental design involves a comparison of the kinetics of a compound formation of the crystal suspension with the kinetics of the soluble material under identical conditions. While this has been possible with ferrimyoglobin because of the slow solubility of the crystals (Chance et al., 1966), the more rapid solubility of the E crystals prevents this approach. Instead we have studied the kinetics of a heterogeneous system in which varying portions of E were in solution or in the crystalline or amorphous form. For example, if the protein obtained as described above is diluted in 2·9 M-ammonium sulfate, about half is in solution and half in
HORSE LIVER ALCOLHOL DEHYDROGENASE
517
the precipitated state at 20°C (Fig. I). If on the other hand, a solution of E is added to 3 M-ammonium sulfate, a portion will be in solution and the remainder either as an amorphous or microcrystalline precipitate; the precipitates obtained in the experiments of Fig. 1 had a crystalline sheen although no crystals could be resolved microscopically.
4. Results (a) Spectroscopic studies Figure 2 illustrates the effect upon the formation of ER of ammonium sulfate concentrations below and above the solubility values for the crystals. In this experiment, 2·4 fLN-crystalline E was added to 2'5, 3·0 and 3·5 M-ammonium sulfate and treated with two equivalents of R (4,8 fLM). From Fig. 2(a}, at 2·5 M-ammonium
J..
325 to 360 mJ1'
~Inereased absorption at 325
17 sec+!
1+
17 see-l
log Joll =0,005 (2 em path)
t
mf'
I-
17 see-J
~
I
I-
\
2'4 fLN-E '-
...
'n.
o.
UJ
~.
t
.
..
I
.di
j
.-
4,.8 j.£M-DPNH
4·8 fLM-DPNH
2·5 M-Ammonium sulfate
3·0 M-Ammonium sulfate
(0)
(b)
..
i
4,8 fLM-DPNH
3·5 M-Ammonium sulfate (c)
FIG. 2. Kinetics and extent of formation of ER at various concentrations of ammonium sulfate. The concentrations of E and R are indicated in the diagram. The ammonium sulfate was buffered with 0·1 Il- phosphate buffer (pH 7,0). A downward deflection of the traces indicates a decrease of absorbanoy at 360 mil- with respect to 325 mil-. The sequential additions of DPNH in (a) and (b) indicate that the absorbancy of DPNH itself is balanced out at the wavelength pair selected. The bottom trace shows time marks every 5 sec. Experiments in an open cuvette of a 2-cm optical path; volume 6 ml.; temperature 25°C. (1216.12, 10, 6 IV.)
sulfate the enzyme is in solution and the addition of two equivalents of R caused an abrupt drop of the trace within the mixing time, while subsequent additions caused no further reaction. The absorbancy calibration indicates the formation of 2·5 fLN-ER. In 3 M-ammonium sulfate (Fig. 2(b)} the addition of two equivalents of R caused a rapid drop, as in the previous experiment, except that the amplitude of the rapid phase was only two thirds of the maximal excursion of the trace; the remaining third of the reaction proceeds in a slower phase with a half-time of about ten seconds. The total amount of ER formed under these conditions is 2·5 fLN. Only 1·6 fLN is formed in the rapid step, just a little over half the total enzyme. In 3·5 M-ammonium sulfate (Fig. 2(c)}, there is no rapid phase of absorbancy change caused by the addition of R, only the slow phase was recorded. Furthermore, the half-time of the slow phase is approximately eleven seconds, comparable to that
518
H. THEORELL, B. CHANCE AND T. YONETANI
of the slow phase in 3 M-ammonium sulfate. The total absorbancy change observed in the reaction is diminished to approximately half the value obtained in the preceding two charts. We have already considered the possibility that the absorption of ER in the crystalline material may be less than that in the solution, but believe that this is unlikely because ofthe thinness of the crystals and the relatively small concentrations of E that they contain (8 ma). Furthermore, the fluorometric data of Fig. 5 suggest that there is no decrease in the amount of ER formation; the amplitude of the fluorescence change is undiminished in the crystalline material. We have therefore, at present, no explanation for the diminished extent of the reaction of Fig. 2(c). However, since our attention here is concentrated upon the kinetic aspects, we are able to compare the rate of reaction of Fig. 2(c) with the rate that would have been observed with the rate of Fig. 2(a) (when measurements with the rapid flow apparatus are then taken into account (Fig. 4)). In summary, a progressive increase of ammonium sulfate concentration results in obliteration of the rapid phase of the reaction and its replacement by a reaction with a half- time of ten seconds. A comparison of the development of the slow phase of the reaction with the salting out curve for the enzyme (see Fig. 1) clearly indicates a relationship between the state of crystallization of the enzyme and its reduced rate of reaction with the coenzyme. If, instead of the crystalline material, amorphous E is added to 3·5 M·ammonium sulfate, the results seen in Fig. 3(a) and (b) are obtained. At 3 M-ammonium sulfate, the addition of 4'8 /kM-R to 1·7 /kN-E caused rapid formation of ER, and addition of a further equivalent of R caused no further reaction. In 3·5 M.ammonium sulfate, addition of 4·8 /kM-R to 2·6 /kN.E caused a rapid absorbancy change corresponding to 1·7 /kN.ER; however, this was followed by a considerably slower reaction. Here we may tentatively attribute the rapid portion of the reaction to the amorphous
I
325 to 360 mJL ,Increased absorption at 325 m/k
1T
log Jo/ l = 0·005 (2 em optical path)
~17 sec
1'4-17 sec
r-l
2'6/-1-N-E
r+
1'7f!'N-E
r
-..j
IV'-
~
~~,
.....
-
...., "I.
U
4'8/kM-DPNH
,
-
....--
--
i\"I "lr1W11~
4·8JLM-DPNH
3·0 M-Ammonium sulfate
3·5 M-Ammonium sulfate
(a)
(b)
FIG. 3. illustrating the effect of ammonium sulfate concentration upon the reactivity of amorphous ADH. Other conditions identical to those of Fig. 2. (1216-9, 8 IV.)
HORSE LIVER ALCOHOL DEHYDROGENASE
519
precipitate of the protein. The slower portion ofthe trace may be due to the formation of a microcrystalline precipitate of extremely low reactivity (see p. 524). (b) Rate of formation of E R
Figure 4 illustrates the high reactivity of E towards R in 2·5 M-ammonium sulfate. This trace has been obtained with the regenerative flow apparatus (Chance, 1964) and the main chamber is filled with ,....,.,1 fLN-horse liver E. The small syringe is filled
1+sao msec RY~4msec --.j
Flow ... velocity
I--
1-
i\
1-
/-- ' -
......... -.'
log 10/1 = 0'002
-
T
t
10fLM-DPNH
FIG. 4. Illustrating the rapid combination of E and R in 2·5 xr-ammonium sulfate. Approximately lIJoN.E mixed with 800 IJoM·R, with 360 IJoM·forlllaldehyde present. The top trace indicates increasing flow velocity of a downward deflection. The maximum deflection corresponds to a time after mixing of 4 msec. The wavelengths are 321 and 360 mu.; temperature 25°C; 0·1 IJo phosphate buffer. (1211·4 IV.)
with 800 fLM-R and since the dilution factor is 80-fold, 10 p.M-R was delivered into the observation tube. Upon initiating the flow, the flow velocity indicator (top trace) indicated a maximal deflection corresponding to a time after mixing of approximately four milliseconds. The spectrophotometric trace (somewhat delayed with respect to the flow velocity trace because ofthe long averaging time employed in order to record the small optical density change (O'002jdivision)) recorded a downward deflection to a decreased level of absorption at 360 mfL and remained roughly constant until the flow stopped, when the larger increment of absorption was observed. The total change corresponded to the formation of 1 fLN-ER. The enzyme intermediate remains in a steady state of turnover activated by the presence of a small (360 fLM) concentration of formaldehyde, until R is exhausted and free E is formed. It is for this reason that the observation chamber was initially filled with free E, and not ER resulting from the previous experiments. The data of this and other experiments indicate approximately half completion of the reaction at four milliseconds. This value may be compared to the half-time of ten seconds for the slow phase of formation of ER in 3 and 3·5 M-ammonium sulfate with 4·8 fLM-R. At equal concentrations of R, the ratio of half-times is about 1000-fold. The velocity constant for the formation of ER calculated from Fig. 4 is about 2 X 107 M -1 sec - \ in better agreement with the value of 1·1 X 107 measured from the over-all reaction (Theorell & McKinley-McKee, 1961) than with that determined in our earlier experiments (Theorell & Chance, 1951). However, the main purpose of this demonstration is to show the high reactivity of the soluble form of E at high ammonium sulfate concentrations similar to those employed in the preceding Figure.
520
H. THEORELL, B. CHANCE AND T. YONETANI
(C) Fluoromeiric studies
Since it is possible that the nature of the ER compounds formed at high concentrations of ammonium sulfate would differ from those formed at lower ionic strength, and also since it is of interest to determine that the R is bound to the crystalline enzyme in such a way as to enhance its fluorescence at the high ammonium sulfate concentrations, we have made some studies of the kinetics of the fluorescence changes under the conditions of Fig. 2. Figure 5(a) to (d) represents a series of titrations of E with R in the presence of 2·9 mx-I, In the absence of ammonium sulfate and the presence of 0·1 M-phosphate, sequential additions of 0·8 JLM-R gave an end point to the titration very close to 1·2 JLN-E.
I
I
-
~'-= '---.
2·9 mM-IBA
I
1-1 min
I
I
I I
2'9 M-Ammonium
su lfate (c)
FIG. 5. Fluorometric measurements of the effect of ammonium sulfate concentration upon the combination of EI and R in the presence of 2·9 mM-I. The concentrations of E and R added are indicated in the Figure 0·1 ,.,. phosphate buffer; 25°C. (1213-13,19,21,24 IV.)
When crystals were added to 0·1 JLM-phosphate buffer, they dissolved immediately; addition of 1·2 JLN-E caused no significant deflection of the trace, nor was there any deflection when 2·9 mM-I was added. The first addition of 0·8 fLM.R gave two thirds of the maximal deflection, and a plateau was reached after three more additions of the coenzyme. The fluorescence increase per 0·8 fLM-E is 3·3 scale divisions. The reaction of R is so rapid that it occurs within the mixing time. In the experiment of Fig. 5(b), 1·5 fLN-crystalline enzyme was added to 2·5 M-ammonium sulfate. The upward deflection of the trace is attributed to the dissolving of the crystals; the crystals caused a considerable scattering of the excitation into the measuring photomultiplier, but as they dissolved, the scattering diminished with a half-time of about ten seconds. The total change of fluorescence due to solution of the enzyme was 2·0 large scale divisions. Addition of I caused no further change, and titration with 0·8 fLM-R caused a rapid increase of fluorescence of 3·3 scale divisions as in Fig. 5(a). The reactions were again so fast that the rate was limited by the mixing time. In the experiment of Fig. 5(c), 2·9 M-ammonium sulfate was employed. The initial addition of E gave a deflection of 2·7 scale divisions. There followed a much slower return of the trace than in Fig. 5(b) and the extent of the return was 1·7 scale divisions.
HORSE LIVER ALCOHOL DEHYDROGENASE
521
At the time of the I and R additions, a portion of the material equivalent to one scale division can be considered to have remained in the crystalline state. The first addition of R gave a rapid deflection of 3·1 scale divisions. The three succeeding additions caused a further fluorescence increase of 2 scale divisions and showed less rapid kinetics than in the previous experiments. In Fig. 5(d) at 3·5 M-ammonium sulfate there was no decrease of deflection following the addition of E. The crystals remain as such under these conditions (see Fig. 1). Addition of 0·8 fLM-R caused a total deflection of 32 divisions, as in the other cases. Now, however, the rapid portion of this trace is almost imperceptible and the halftime ofthe reaction is about 20 seconds. Further additions ofR complete the titration, but in each case it is apparent that a slow change was involved. It is appropriate to compare the results of Fig. 5(d) with those of Fig. 2(c). The slower rate in Fig. 5(d) is due to the lower concentrations of both E and R employed in the fluorometric experiment. Thus, the reactivity of the crystalline material is very small whether measured spectrophotometrically or fluorometrically. (d) Reactivity towards isobutyramide
While the data of Figs 2 and 5 demonstrate the low activity of the crystalline enzyme towards R, the reaction ofER and I, a smaller molecule, is presented in Fi~. 6. In Fig. 6 we have employed the fluorometric technique of Fig. 5 and the ER compound is formed from 1·2 fLN-E and 2·4 fLM-R in 3·5 M-ammonium sulfate. The trace then shows the increase of fluorescence caused by the addition of 2·9 mM-I to ER. An increase of fluorescence (4 scale divisions) similar to the fluorescence increase of Fig. 5(d) is seen, where R is added to EI. The rate appears, however, to be more rapid; the first three divisions of deflection occur in the mixing time and only the last 20% occurs in a measurable slow reaction.
T
0·8 fLN-ERI
1 FIG. 6. Fluorometric determination of the rate of combination of 1·2 I"N-ER with 2·9 mM-I in 3·5 msr-ammonium sulfate. 0·11" phosphate buffer (pH 7'0); temperature 25°C. (1213-17 IV.)
It is difficult to compare this reaction with the data of Fig. 5. The ratio of I to R employed in the two experiments is approximately 3000:1 and thus a fairly rapid reaction would be expected in Fig. 6. While somewhat lower concentrations of I can be used, the larger dissociation constant for this substance prevents its use at concentrations of the same order as employed in the reaction of EI plus R. Figure 6 does demonstrate that even with as high a concentration of I as 2·9 mx, a portion of the reaction requires more than a minute for its completion.
H. THEORELL, B. CHANCE AND T. YONETANI
522
(c) Reaction of 0 with the enzyme-pyrazole compound
Figure 7 shows a series of traces at increasing ammonium sulfate concentrations, similar to those of Figs 2 and 5. Here the reaction of the enzyme-pyrazole compound (EP) with 0 is recorded in terms of the increased absorbancy change at 325 mfL, using 395 mfL as the reference wavelength (Theorell & Yonetani, 1963). In trace (a), 295 to 325 mfL Increased absorption at 295 mIL optical path 2 em
',4
fLJ1Mj-j~PN 2.8_ .-
r
.+
jiM_DPN
~ 2'8jtM-DPN
log 1011= 0·01
-+~
IC!'4~Eti T~,!!o$ log 101[= 0-\1
r- T -l ~~c I+L-
No ammonium sulfate (a)
=t=t==
-_-+j sec 60 I+-_ I I
2·5 M-Ammonium sulfate ( b)
t I fL
I
M -DPN
i'
.if--'·8 I~ I log [011-0'01 r---
~
60 _ -+j sec I+-_
I
I
3·0 M-Ammonium sulfate (c)
3·5 M-Ammonium sulfate (d)
FIG. 7. Spectrophotometric determinations of the reactivity of EP with 0; 1·4 mM.P; concentrations of E and 0 indicated in the Figure. A downward deflection indicates increased absorbtion at 295 mu relative to 329 tau, 0·1 ,.,. phosphate buffer (pH 7'0); temperature 25°C. (1216A, 3, 14, 9,10 IV.)
several portions of 1·4 fLM-O are added to 1·2 fLN-E. The abrupt downward deflection of the trace indicates the combination time in the absence of ammonium sulfate to be less than the mixing time. More than 80% of the maximal deflection is obtained with one equivalent of 0, and the total absorbancy change corresponds closely with the formation of 1·2 fLN-EPO. In trace (b) there is a single addition of 2·8 fLM-O to 2·4 fLN-enzyme in 2·5 M-ammonium sulfate. In this case, the rate of formation of the EPO compound is readily recorded, and the reaction occurs at a measurable rate with soluble enzyme in 2·5 M-ammonium sulfate. It is possible that sulfate itself has inhibited the reaction somewhat. At 3·0 M-ammonium sulfate (Fig. 7(c)) the reaction is somewhat slower and the extent is smaller. Finally, at 3·5 M.ammonium sulfate (Fig. 7(d)), where the enzyme is all in the crystalline form, the amplitude is further reduced and the rate further slowed. There is some evidence under these conditions that the reaction is biphasic, a feature not observed in the kinetics of Figs 2 and 5. In summary, we find the reaction of EP with 0 to be quite slow in 3·5 M-ammonium sulfate.
5. Discussion The striking effect of a crystallization upon the speed of combination of ADH with DPNH as measured spectrophotometrically or fluorometrically in the presence of I supports and greatly increases the generality of the conclusions based upon studies of a suspension of ferrimyoglobin crystals with azide (Chance et al., 1966). In spite of the fact that, in the case ofE, it was not possible to measure completely crystalline
HORSE LIVER ALCOHOL DEHYDROGENASE
523
and completely soluble materials at the same concentrations of ammonium sulfate, studies of a gradation of effects with increased ammonium sulfate follow the "salting out" curve for the enzyme so closely that the decrease of reactivity of E with increasing ammonium sulfate concentration can be attributed to the preservation of crystal form at the higher concentrations. The extent of decrease of reactivity of E can be quantitated for the reaction with R; the ratio of the half-times of the reaction of E with equal concentrations of R at 3·5 and 2·5 M-ammonium sulfate is in excess of 1000. The ratio of the half-times in the reaction with 0 in the presence of pyrazole at 3·5 and 2·5 M.ammonium sulfate is about tenfold. This is a surprising result, because EIR and EPO are crystallographically isomorphic, and one would have expected that the steric and other factors involved in their formation in the crystal state would have been identical. As a matter of fact, the half times of EIR and EPO formation are not greatly different at 3·5 M-ammonium sulfate. However, the fact that the EP 0 reaction proceeds at a measurable velocity at 2·5 M-ammonium sulfate suggests that another factor is operative in this case. A possible explanation is afforded by the existence of an induction period in the formation of the pyrazole complex which is absent in the case of the R reaction of E and I (Theorell & Yonetani, 1963). Many factors involved in the binding of the enzyme and coenzyme could be altered on crystallization, and thereby provide an explanation of the decreased reactivity of the crystalline material. Diffusion ofthe coenzyme through a water phase to the active site appears unlikely to be a significant factor in the decreased reactivity of the crystalline material. The existence of a water phase in the crystal is indicated by the observation of 48% water content in the unit cell. The diminution ofthe R concentration due to combined diffusion and reaction velocity in this water phase can be calculated for the thin plates of E by an equation which is considerably simplified compared to that used for the calculation of the cylindrical geometry of the ferrimyoglobin crystals (Roughton, 1932). (Thanks are due to Dr R. Foster for assistance in the diffusion calculations.) The ratio of the R concentration in the crystal and in the surrounding solution is given by the following expression, using the terminology of Appendix I (Chance et al., 1966): [R] mean inside 1 [R] at surface
+
!-JkE
2])
where t is the thickness of the crystal; k, the second-order velocity constant for the combination ofE and R; E, the enzyme concentration; and D, the diffusion coefficient of R. Since most of the crystals were of a thickness of 11-' or less, tj2 is 5 X 10 - 5 em, The second-order velocity constant is taken to be 1 Xl0 7 M- 1 sec- 1 (see above). The concentration of E in the crystals is approximately 8 mM. The diffusibility of R is decreased compared to azide by the square root of the ratio of the molecular weights (-\/17) or 2·5 X 10- 6 cm 2jsec. A substitution of these values into the equation indicates that the azide concentration within the plates is 0·12 that at its surface. It is apparent that the thinness of the plates and the low concentration of E in the crystals compensates for the high reaction velocity constant of combination and the low diffusibility of R. Consequently, the conditions for observing the reaction of E and R in crystals of E without interference from diffusion are only fourfold less
524
H. THEORELL, B. CHANCE AND T. YONETANI
favorable than those for the observation of the reaction of ferrimyoglobin and azide. It is of great interest that the combined reaction and diffusion equation fails much more seriously in this case to explain the slowness of the observed reaction of E and R; if, indeed, the equation afforded an explanation, we would at least expect a proportionality between the calculated ratios of concentrations (inside and outside) and the observed values of k. This is not the case; for azide, the ratio is 0·5 x21 =11, and for this case the ratio is 0·12 X1000=120. Thus, the "fit" is ten times worse for the reaction of E and R. Since we may conclude that k is decreased, the ratios of inside to outside oonoentrations must be even nearer unity, or the tolerance of error of the diffusion constant much greater. For example, for k = 3x104 M- 1 seo" ", D may fall to 2·5Xl0- 8 cm 2/sec to give the same inside-outside ratio calculated above. The possibility that the crystals are completely unreactive and that the reactivity observed is due to the rate of solution and recrystallization of the enzyme is to be considered. The experimental data suggest, however, that this is not the principal factor in these particular circumstances. In Fig. 2(b) and (0), where the ammonium sulfate concentration is increased from 3·0 to 3,5, the half-time for the slow phase is not greatly decreased. However, Fig. I shows that these two sulfate concentrations would very greatly decrease the amount of free enzyme in equilibrium with the crystals. The possibility that the crystals may have become coated with a monolayer of protein impurity has also to be considered. The slow portion of the kinetics of Fig. 3 is helpful on this point, and suggests that a microcrystalline material of a very large surface-to-volume ratio became "coated" to the same extent as the larger crystals of Fig. l(c), which we regard as very unlikely. REFERENCES Blake, C. C. F., Koenig, D. F., Mair, G. A., North, A. C. T., Phillips, D. C. & Sanna, V. R. (1965). Natvre, 206,757. Boyer, P. & Theorell, H. (1956). Acta Ohem, Scand. 10, 447. Branden, C. 1. (1965). Archiv. Biochem, Biophys. 112, 215. Chance, B. (1951). Rev. Sci. Lnstr., 22, 619. Chance, B. (1952). In Modern Trends in Physiology and Biochemistry, ed. by E. S. G. Baron, p. 25. New York: Academic Press. Chance, B. (1964). In IUB/IUBS Rapid Mixing and Sampling Techniques in Biochemistry, ed. by B. Chance, Q. H. Gibson, R. Eisenhardt & K. K. Lonberg-Holm, p. 125. New York: Academic Press. Chance, B., Ravilly, A. & Rumen, N. (1966). J. Mol. Biol. 17, 525. Dalziel, K. (1961). Biochem, J. 80, 440. Keilin, D. & Mann, T. (1936). Proc, Roy. Soc. B, 122, 119. Kendrew, J. C., Dickerson, R. E., Strandberg, B. E., Hart, R. G., Davies, D. R., Phillips, D. C. & Shore, V. C. (1960). Nature, 185, 422. Nobbs, C. L. (1965). J. Mol. Biol. 13, 325. Perutz, M. F. (1965). J. Mol. Biol. 13, 646. Roughton, F. J. W. (1932). Proc. Roy. Soc. B, Ill, 1. Stryer, L., Kendrew, J. C. & Watson, H. C. (1964). J. Mol. Biol. 8, 96. Theorell, H. (1942). Enzymologia Acta Biocatalytica, 10, 250. Theorell, H. (1962). Pontijicae Academiae Scientiarum Scripta Varia, 22, 1. Theorell, H. & Bonnichsen, R. (1951). Acta Ohem, Scand. 5, 1l05. Theorell, H. & Chance, B. (1951). Acta Ohern; Scand. 5, 1127. Theorell, H. & McKinley-McKee. (1961). Acta, Ohem. Scand. 151797. Theorell, H. & Yonetani, T. (1963). Biochem, Zeit. 338, 537.