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The α-chymotrypsin catalyzed hydrolysis of ethyl lactate
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
76, 148-160 (1958)
The a-Chymotrypsin Catalyzed of Ethyl Lactate
Hydrolysis
Ignacio Tinoco, Jr. From the Department
of Chemistry, Received
University October
of California,
Berkeley,
California
27, 1957
INTRODUCTION The catalytic activity of cr-chymotrypsin has been studied very extensively (l-8). Most of the nonprotein substrates have been compounds of tyrosine, tryptophan, phenylalanine, or analogous structures (l-5) ; some measurements have been made on substituted phenyl esters of n-paraffin acids (6-g). However, it was felt that a study using an even simpler substrate might be more amenable to interpretation in terms of electrostatic, or other, specific interactions. Ethyl lactate was chosen as the substrate because its hydrolysis is catalyzed by chymotrypsin at an easily measurable rate, it is completely miscible with water, and it is commonly available. Measurements of Kz and A$ in the simple Michaelis formulation :
0) were made over the pH range 5.2-8.6 in 0.1 M NaCl at 25°C. No buffer was used because in order to study a wide pH range, different buffers are often needed, and even with one buffer the ratios of the components change with pH. These different buffer constituents are often inhibitors or activators of enzymes and might thus mask the primary pH effect. EXPERIMENTAL Materials The or-chymotrypsin was a crystalline, salt-free product (Lot No. CD 514-17) obtained from Worthington Biochemical Co. To remove any insoluble matter from the enzyme, the Worthington chymotrypsin was dissolved in water, filtered through an ultrafine sintered-glass filter, and lyophilised. Most of the experiments 148
WCHYMOTRYPSIN-CATALYZED
HYDROLYSIS
149
were done with this preparation; however, the same results were obtained with the original material. Various sources of ethyl m-lactate were used. Most of the experiments were done with Eastman White Label grade; for some experiments the ethyl lactate was distilled through a 30-plate column at 1 cm. pressure. Preliminary experiments were made with a Mathieson, Coleman, and Bell sample. There was no difference in the kinetic behavior of these different samples. It was noted, however, that ethyl lactate once exposed to the air underwent a slow change over a period of months. Ethyl lactate which had been in a previously opened bottle for 3 months gave a lower pH when dissolved in water and hydrolyzed faster (with either base or chymotrypsin catalysis) than a fresh sample of ethyl lactate. Apparently slight hydrolysis occurred followed by reaction of the acid with the a-hydroxyl, leading to lactyl lactate and possibly higher polymers. The concentration of polyester was small because with continued hydrolysis the rate decreased and reached a value equal to that of fresh ethyl lactate. For all the experiments reported, the ethyl lactate was from recently opened bottles and gave a constant rate of hydrolysis . Reagent-grade NaCl and boiled distilled water were used in preparing all solutions.
Methods The rate of hydrolysis of ethyl lactate was measured by continuously titrating the lactic acid released. NaOH (0.1011 N) was delivered into the reaction beaker by a motor-driven Gilmont microburet. The Bodine d.c. variable speed motor was turned on or off by an amplified’ signal from a Beckman model G pH meter with external electrodes. Whenever the pH dropped below a predetermined value, base was added from the buret to keep the pH constant. The speed of the buret drive could be varied continuously from 0.033 to 45 r.p.m. by variation of the voltage to the motor, plus the use of three gears; this corresponds to a delivery rate of from 0.001 to 1.35 ml./min. with a l-ml. plunger in the buret. The pH could be kept constant to f0.05 or less with the sensitivity increasing for the intermediate rates. The volume of base added and the time were recorded manually. The reaction beaker was immersed in a constant-temperature water bath at 25.00 f .05”C. The solution in the beaker was stirred by means of a waterproofed magnetic stirrer in the bath. Nitrogen was blown over the top of the reacting solution to exclude CO2 ; in order to avoid evaporation of this solution, the nitrogen was first bubbled through a solution of approximately the same composition. The beaker was covered by a loose-fitting cap. Reaction solutions were made by mixing ethyl lactate, 1 M NaCl, and water to give a total volume of 19 ml. The rate of hydrolysis was measured for about half an hour; then 1 ml. of the enzyme solution was added, and the rate was measured for another half hour. In a few experiments the rate was measured for times up to 2 hr. The enzyme solution was made daily by dissolving the chymotrypsin in a known volume of water. The concentration was calculated from the weight of the 1 We wish to thank of the amplifier.
Professor
R. E. Powell
and Mr. H. Robinson
for the design
150
TINOCO, JR.
enzyme used and a knowledge of its water content, which was determined by drying it in vacuum at 11O’C. The concentration of enzyme in the reacting solution was 1 g./l. The hydrolysis was pseudo-zero order under the conditions used. The rate was slow so that usually only a small fraction (1%) of the ethyl lactate was hydrolyzed; the pH and the concentrations of enzyme and water were also constant, except for a slight dilution of the solutes during the reaction by the addition of the base. The total dilution was always less than 5% and often less than 1%. Rates were calculated from the slopes of plots of base volume vs. time. The plots were consistently linear. In the calculation of rate constants, the average concentrations during the reaction were used. At each pH about ten different substrate concentrations, covering the range from 0.02 to 1 M, were studied. The rate in the absence of the enzyme (which includes effects of CO, absorption) was plotted vs. substrate concentration. The appropriate rate to subtract from the rate in the presence of the enzyme could then be obtained. The rate constants k, (sec.-l X 107) for these non-enzymic hydrolyses [k, = -(dS/dt)(l/S)] were: pH 8.6, 60.2; pH 8.2, 27.2; pH 7.8, 11.3; pH 7.4, 5.92; pH 7, 3.14. For the experiments at pH’s 5.2, 6.0, and 6.5 the non-enzymic rates were obtained by linear extrapolation from the rates at higher pH’s. At the lower pH’s the non-enzymic rates were never greater than 20% of the enzyme-catalyzed rates. For one experiment a sample of diisopropyl phosphoryl (DIP) chymotrypsin was used. It was prepared by treating chymotrypsin in 0.2 M phosphate buffer at pH 7.3 with a tenfold excess of diisopropyl phosphorofluoridate (diisopropyl fluorophosphate) (DFP).* After 4 hr. at room temperature, the solution was dialyzed vs. water at 5°C. RESULTS
The kinetic parametersa ki and Ki were obtained at each pH from a least-squares treatment of Lineweaver-Burk (9) plots (Figs. l-3). From the linearity of the plots it is apparent that there is no substrate inhibition over the 50-fold concentration range studied. An exception is pH 8.6 where there is definite indication of substrate inhibition above 0.5 M. The results are given in Table I, with probable errors (10) for all values. For the calculation of ki a value of 25,000 was used for the molecular weight of chymotrypsin. There is a wide variation in the reported values of molecular weight (11-13) ; this one, based on analytical results, was chosen because hydrodynamic and light-scattering measurements are greatly affected by the well-known polymerization of chymotrypsin. In the study of a new substrate it is important to show that the * We wish to thank Dr. Irving Geschwind and the Hormone Research Laboratory of the University of California for help with this preparation. a See the Appendix for the relation between these parameters for a nn-mixture and the individual values for the D- and n-isomers.
2.5
2.0
I.5 I/V x IO' (M:'Sac.) 1.0
I
I
I
I
IO
20
30
40
I/S (M.-‘) hydrolysis of 1. Lineweaver-Burk plots for the m-chymotrypsin-catalyzed ethyl DL-lactate at pH’s 6.2, 6.0, and 6.5 in 0.10 M NaCl at 25%. The scale of the ordinate for the pH 5.2 data has been divided by ten. FIG.
.6
FIG.
2. Lineweaver-Burk of ethyl nL-lactate
plots for the a-chymotrypsin-catalyzed at pH’s 7.0, 7.4, and 7.8 in 0.10 M NaCl 151
hydrolysis at 25°C.
152
TINOCO,
JR.
I/Vx IO’ WSec.) I
I/S th4.-‘I
FIG. 3. Lineweaver-Burk of ethyl DL-lactate
plots for the a-chymotrypsin-catalyzed at pH’s 8.2 and 8.6 in 0.10 M NaCl TABLE
Kinetic
Parameters
PH
Hydrolysis
of Ethyl
in 0.10 M NaCE at WC.
.q x 10’ (sec.-l)
0.20 0.78 2.2 2.78 3.85 3.9 3.1 2.8
hydrolysis at 25°C.
I
for the a-Chymotrypsin-Catalyzed Dr.-Lactate
5.2 6.0 6.5 7.0 7.4 7.8 8.2 8.6
&--- &- -z-k-
f f f f f f f f
.04 .1 .2 .l .l .3 .5 .6
K:(M)
0.46 0.20 0.25 0.14 0.16 0.12 0.10 0.11
f f f f f f f f
0.1 .02 .03 .Ol .Ol .Ol .02 .02
apparent activity of the enzyme is not caused by other trace enzymes in the preparation. Competitive inhibition of the new activity by a specific inhibitor is good evidence for the identity of the enzyme. At pH 7.4 @phenylpropionate was studied as an inhibitor for the ethyl lactate activity. Measurements at six substrate concentrations in 0.02 M /3phenylpropionate gave a competitive inhibition constant of 0.003 M. This is in reasonable agreement with values of approximately 0.005 M
CX-CHYMOTRYPSIN-CATALYZED
153
HYDROLYSIS
obtained at pH 7.9 by Neurath et al. (14) with acetyl-L-tyrosinamide as a substrate, and thus shows that chymotrypsin is responsible for the ethyl lactate activity. It is possible, however, that some specific inhibitors of chymotrypsin would not affect this activity. For example, phenol, which is a competitive inhibitor for chymotrypsin substrates (15)) might not interfere appreciably with a chymotrypsin-ethyl lactate complex. It is also of interest to determine whether other groups on the enzyme, besides those on the catalytic site, contribute appreciably to the hydrolysis of the substrate. DIP chymotrypsin had no catalytic activity for ethyl lactate at pH 7.4. This confirms the catalysis as due to the specific esterase site of the enzyme, and differentiates ethyl lactate from the phenyl esters as substrates. Phenyl esters are hydrolyzed, though more slowly, by DIP chymotrypsin (7). This residual activity of inhibited chymotrypsin is apparently due to catalysis by the other imidazole group in the molecule (16, 17). DISCUSSION
Recent studies (8, 18, 19) have shown the need for modifying the classical mechanism for chymotrypsin action. We may write the following equation incorporating these new ideas: E+S+
ES1 -
kz
1
ES2 +p,
k,
ESs -
kh
E + Pz
(2)
‘k--a
The various enzyme-substrate complexes are: ES1 , the Michaelis complex; ES2, the serine hydroxyl-acylated complex; and ES1, the histidine imidazole-acylated complex. The products are PZ , the acid part of the substrate; and PI, the alcohol, amide, etc., portion. The symbols in Eq. (2) refer to all the ionized forms of substrates or products at a given pH, i.e., the stoichiometric concentrations. We need not consider steps involving kL2 and kL for the initial stages of the reaction when the concentrations of products are small. At a constant pH the rate of formation of products in the steady state is given by the following equations : dP1 -= dt 1 -= k;
dPz -= dt 1 1 a+k,+lc,
k%E) 1 + G/(S) 1
K~ = (k-1 + k&i 23 klkz
(3)
154
TINOCO,
JR.
The Michaelis form is retained, but the parameters have a different significance. One sees that ki is a measure of the rate-determining step, if there is one. Furthermore, Ki is in general proportional to ki. A discussion of these equations separates naturally into two parts, depending on whether kz is or is not rate determining. If kz is rate determining, we have the classical result: (Class 1)
k; = kz ;
Ki = (k-l + kZ)/kl
(6)
The step following the formation of the enzyme-substrate complex is rate determining, and Ki is an equilibrium dissociation constant if kV1 >> kz . If kz is not rate determining we have the following more complicated equations: (Class 2)
Ic4 leoa= 1 + (k-g + k.,)/ks (k--l + kz)k4 K’ = klkz[l + (k-3 + kJ/ks]
(7)
The rate is controlled by the transfer of the acyl group from the serine oxygen to the imidazole nitrogen, and its subsequent hydrolysis. pH Dependence The pH dependence of ki has often been used to determine the pK’s of groups involved in the catalytic site of the enzyme (3, 5, 8, 20) One assumes that the enzyme site is active only if it is in a certain ionized form, and that the rate of reaction of this special ionized form is pH independent. These assumptions lead to the following equations for the Michaelis-Menten formulation if one or two ionizing groups, respectively, are involved (21, 22). k: = k; =
i&o 1 + (H+>/Ko ki,o 1 + (H+)IK,
+ Ka/(H+)
For the mechanism given in Eq. (2), we must examine the pH dependence of each rate constant. There is no direct evidence about kl and k-1 ; for specific substrates where most of the binding is through
a-CHYMOTRYPSIN-CATALYZED
HYDROLYSIS
155
an aromatic ring we would expect these rate constants to be insensitive to pH, however. The pH dependence of kz is given by (8, 23):
group in where K,, 1 is the ionization constant of the imidazolium ES1 . Studies of reactions between an ester and free imidazole (16, 17) have shown that kl should have the same type of dependence:
Within the framework of the present (21,22) theories of pH dependence, one can show that Eq. (9u) requires that k-3 obey the following relation:
However, for simplicity we shall assume that k--l is pH independent in the pH range of interest. Comparing the relative stabilities of imidazolium ion (in E&) and N-acylimidazolium ion (in E&), one would expect Km,3 >> lOK,,z . This makes the pH dependence of k--) slight in the pH range where kS is important. In effect we have given the equilibrium constant (k,/kJ for the 0-acyl ti N-acyl equilibrium the type of pH dependence usually given the individual rate constants. The 0-acyl e N-acyl migrations are well known in amino alcohols (24, 25), and the pH dependence of the equilibrium agrees qualitatively with that postulated above. The rate constant kr for the hydrolysis of the acyliiidazole must have the form: k4 = k+(H+)
+ k4.w (6.0) + k4.b (OH-)
(10)
This equation usually implies that k4 will pass through a minimum in neutral solution and increase in both acid and basic solution. As no chymotrypsinor imidazole-catalyzed hydrolyses show this pH dependence, we conclude that either k4,w is very large or k4 is not rate determining. In either case we can safely take kc as pH independent. As the pH dependence of Class 1 has been described many times before (21, 22), it will not be discussed further.
156
TINOCO,
Class 2, however, is of interest. reads : (Class 2)
”
= 1 + (k-s +
kr)(l
JR.
Our pH-dependent
equation
k4 + H+/K,,z)/ks,o
now
(11)
or k4
1 + (k-3 +
k4)/ks,o
I[
1
1 + H+/Ka,z[l + ko/(k--3 +
k4)l
1(114
One sees that we have the same pH dependence given by Eqs. (8) or (9), but the ionization constant as usually determined (8, 21, 22) is an apparent one equal to K,,Jl -I- k~,~/(k-~ + k4)]. Dixon and Neurath (23) and Gutfreund and Sturtevant (8) have found that ~K,J is less than the apparent pK,,z by about 0.6 unit. Whether this change is caused mainly by the change of the ionization constant or by the effect of the three rate constants is not known. Comparing the data for ki vs. pH (Table I) with Eq. (8b), one finds values of pK, = 6.7 and pKb = 8.7 for the ethyl lactatechymotrypsin system. Whether these values refer to ES1 , or ES* and whether they are apparent values which contain [l + /~3,~/(k--3 + Ic4)] depends on the rate determining step. This question will be discussed in the next section. The prediction of the pH dependence of Ki will in general be difficult. However, as the effects of changes in IcZand k3 with pH are in opposite directions, Kg may be nearly independent of pH. It has been claimed that if h$ and Ki have a different pH behavior, then Ki is an.equilibrium constant (3, 20). This argument is clearly not valid for Class 2. Rate Determining Step Information to allow one to choose between Class 1 and Class 2 has been obtained by Gutfreund and Sturtevant (8, 18) using fast reaction techniques. Their mechanism is similar to Eq. (2) except that step 3 is absent. They have found by direct measurements that kz is not rate determining for p-nitrophenyl acetate (Class 2), and they have inferred that kz is rate determining for acetyl-L-tyrosinamide (Class 1). Dixon and Neurath (23) have obtained the same results for p-nitrophenyl acetate, and they have inferred that acetyl-L-tyrosine ethyl ester also belongs to Class 2. A comparison of the kinetic parameters obtained by various investi-
a-CHYMOTRYPSIN-CATALYZED
TABLE A Comparison
of Kinetic
Parameters Solvent
Substrate
Ethyl m-lactate Methyl hydrocinnamatt
Water
2OTo(w/w
157
HYDROLYSIS
II
for a-Chymotrypsin-Catalyzed k :,.
bec.-l)
0.04b 0.026’
LI
b
Hydrolyses
PKZ~
K:(@
6.7 7.2
0.46-0.11 (3.84.1) x 10-z
Ref.
methanol Acetyl-r-tyrosinamide
20%
(v/v)
(3)
6.7
0.045-0.18
(8)
7.3
-
(18)
2-propan01 p-Nitrophenyl
acetate
Acetyl-L-tyrosine ethyl ester Acetyl-L-tryptophan ethyl ester Acetyl-L-phenylalanine
20% (v/v) a-propan01 Water
0.028
190
6.7
Water
52
6.7
Water
150
6.85
7 x 10-t 8.9 x 10-G (1.1-3.1)
x 10-s
(5) (5) (19)
-
D Assuming a mol. wt. of 25,000 for chymotrypsin. b Maximum value. c Apparent value; see text. d The values given correspond to the lowest and highest pH’s does not pass through a maximum or minimum, these represent values.
studied. As Ki the
extreme
gators from studies of pH dependence is given in Table II. If we accept the above conclusions, p-nitrophenyl acetate and the specific amino acid ester substrates are in Class 2, while the amide substrate is in Class 1. To help place ethyl lactate and methyl hydrocinnamate we notice that the pH dependence of these two substrates passes through a definite maximum at pH 7.8, while for the last three substrates the catalytic activity is nearly constant between pH 8 and 9. While the pH dependence of h$ for acetyl-n-tyrosinamide has not been studied above pH 8, measurements at one substrate concentration show a decrease in catalytic activity above pH 8 (26). We would therefore designate the first three substrates in the table as Class 1 and the remaining ones as Class 2. That is, we think the formation of the lactylserine oxygen bond is rate determining for the ethyl lactate hydrolysis and that Ki for this system is the equilibrium dissociation constant for the Michaelis complex. The pK’s of 6.7 and 8.7 therefore refer to
158
TINOCO, JR.
ES1 ; the lower pK, must be for the active imidazolium group. As there is no known group in the chymotrypsin catalytic site with a pK = 8.7, it might be due to a more generalized pH-dependent process in the enzyme which would affect step 2 but not step 3. It is interesting that there is a large change in the optical activity of chymotrypsin at this PH (27). The variation of the pK,‘s for the different substrates is difficult to interpret because of the various solvents used and the effect of differences in i&,0/@--3 -I- k4) on the apparent pK,‘s obtained from Class 2 kinetics. The pH dependence of Kj will in general also be difficult to interpret. The magnitude of Ki for ethyl lactate, however, is of interest because it is much greater than that for other substrates. Comparing ethyl lactate with methyl hydrocinnamate, which has a similar value of ki, we see a 25- to a loo-fold difference in Ki . This difference mirrors the weaker binding between ethyl lactate and chymotrypsin. The fact that Kl for methyl hydrocinnamate is pH independent within experimental error, while Ki for ethyl lactate varies with pH, is probably caused by weak interaction of the cu-hydroxyl with some ionizable group on the enzyme. ACKNOWLEDGMENTS Dr. Susan Lowey and Mr. Donald Crothers made preliminary measurements on this system. We are very grateful for their help. We wish to thank Dr. M. P. Freeman and Professor R. J. Myers for advice on the construction of the pH-stat. We are indebted to Dr. I. I. Geschwind and Professors B. H. Mahan, H. Rapoport, and A. Streitweiser, Jr., for helpful discussion and suggestions. CONCLUSIONS
In the above discussion it has been assumed that there is a ratedetermining step; that is, that an enzyme-substrate system could be assigned to one or another class over the entire pH range. Unfortunately, even at one pH all rate constants can be of the same order of magnitude, and in general as the rate coefhcients vary with pH it is possible that different steps will beoome rate determining. However, kz and k3 have approximately the same pH dependence so that this is not very likely. We must conclude, however, that all decisions about which steps are rate limiting are tentative, except for p-nitrophenyl acetate, where the individual processes have been measured directly. That this deci-
&CHYMOTRYPSIN-CATALYZED
sion is an important one is obvious, Ki and the pH behavior depends on this information include the study of (28) and the use of product inhibition of the substrate.
HYDROLYSIS
159
as the precise interpretation of it. Other methods for obtaining different esters of the same acid by either the acid or alcohol part
SUMMARY
The pH dependence (pH 5.2-8.6) of ki and Ki for the cu-chymotrypsin-catalyzed hydrolysis of ethyl m-lactate in 0.10 M NaCl at 25°C. has been determined. The value of ki first increases with pH; reaches a maximum of 0.039 sec.-l at pH 7.8; then, decreases. The value of Ki decreases from 0.46 to 0.11 M with increasing pH. The kinetics have been discussed in terms of a mechanism involving three enzyme-substrate complexes. It has been tentatively eoncluded that the rate-determining step for ethyl lactate is the ester interchange leading to the formation of enzyme-serine lactate and the release of ethanol. A value of pK, = 6.7 was obtained for the active imidazolium group to which the lactyl is transferred before hydrolysis. APPENDIX As ethyl lactate lacks binding of substrates by point of a stereospecific L-forms of ethyl lactate
the aromatic group which is primarily chymotrypsin, and which is supposed contact with the enzyme, we would to have very similar kinetic behavior,
kZ,DI,E &I
Lz I& ;
responsible for the to provide the third expect the D- and i.e.,
K:.DL S K~.D E K~.L
However, the general relations among the various constants are:
k:.DL.= ~/G.DL.
=
&L + (K~,,/K~,D)ki,D 1 + (K~,LIK~.~)
(Al)
fb)(llK~,~,
(A21
+
~/K:,D)
The pH dependence of each step should be identical for the optical isomers according to present theory; therefore, even if k:,n is not equal to 7c:,n , the same value of pK. should be obtained. REFERENCES 1. GREEN, N. M., AND NEURATH, H., in “The Proteins” (Neurath, H., and Bailey, K., eds.), Vol. II, Pt. B, p. 1057. Academic Press Inc., New York, 1954. 2. FOSTER, R. J., AND NIEMANN, C., J. Am. Chem. Sot. 77, 1886 (1955). 3. LAIDLER, K. J., AND BARNARD, M. L., Trans. Faraday Sot. 62, 497 (1956).
160 4. 5. 6. 7. 8. 9. 10. 11.
12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.
TINOCO,
JR.
BENDER, M. L., AND KEMP, K. C., J. Am. Chem. Sot. 79, 116 (1957). CUNNINGHAM, L. W., AND BROWN, C. S., J. Biol. Chem. 221, 287 (1956). HOFSTEE, B. H. J., B&him. et Biophys. Acta 24, 211 (1957). HARTLEY, B. S., AND KILBEY, B. A., Biochem. J. 66, 288 (1954). GUTFREUND, H., AND STURTEVANT, J. M., Proc. Natl. Acad. Sci. U. S. 43, 719 (1956). LINEWEAVER, H., AND BURK, D., J. Am. Chem. Sot. 67, 658 (1934). of Physics and ChemMARGENAU, H., AND MURPHY, G. M., “The Mathematics istry,” p. 502. D. Van Nostrand, New York, 1943. GREEN, N. M., AND NEURATH, H, in “The Proteins” (Neurath, H., and Bailey, K., eds.), Vol. II, Pt. B, p. 1072. Academic Press Inc., New York, 1954. STEINER, R. F., Arch. Biochem. Biophys. 63, 457 (1954). TINOCO, I., JR., Arch. Biochcm. Biophys. 68, 367 (1957). NEURATH, H., GLADNER, J. A., AND DEMARIA, G., J. Biol. Chem. 188, 407 (1951). HUANG, H. T., AND NIEMANN, C., J. Am. Chem. Sot. ‘76, 1395 (1953). BRUICE, T. C., AND SCHMIR, G. C., J. Am. Chem. Sot. 79, 1663 (1957). BENDER, M. L., AND TURNQUIST, B. W., J. Am. Chem. Sot. 79, 1652 (1957). GUTFREUND, H., AND STURTEVANT, J. M., Biochem. J. 63, 656 (1956). DIXON, G. H., AND NEURATH, H., J. Am. Chem. Sot. 79, 4558 (1957). HAMMOND, B. R., AND GUTFREUND, H., Biochem. J. 61, 187 (1955). ALBERTY, R. A., J. Cellular Comp. Physiol. 47, 245 (1956). LAIDLER, K. J., Trans. Faraday Sot. 61, 528, 540, 550 (1955). DIXON, G. H., AND NEURATH, H., J. Biol. Chem. 226, 1049 (1957). VAN TAMELEN, E., J. Am. Chem. Sot. 73, 5773 (1951). FODOR, G., AND KISS, J., J. Chem. Sot. 1962, 1589. THOMAS, D. W., MACALLISTER, R. V., AND NIEMANN, C., J. Am. Chem. Ser. 73, 1548 (1951). RUPLEY, J. A., DREYER, W. J., ANU NEURATH, H., Biochim. et Biophys. Acta 18, 162 (1955). HOFSTEE, B. H. J., Abstracts Pacific Slope Biochemical Conference, 1957.
OF
BIOCHEMISTRY
AND
BIOPHYSICS
76, 148-160 (1958)
The a-Chymotrypsin Catalyzed of Ethyl Lactate
Hydrolysis
Ignacio Tinoco, Jr. From the Department
of Chemistry, Received
University October
of California,
Berkeley,
California
27, 1957
INTRODUCTION The catalytic activity of cr-chymotrypsin has been studied very extensively (l-8). Most of the nonprotein substrates have been compounds of tyrosine, tryptophan, phenylalanine, or analogous structures (l-5) ; some measurements have been made on substituted phenyl esters of n-paraffin acids (6-g). However, it was felt that a study using an even simpler substrate might be more amenable to interpretation in terms of electrostatic, or other, specific interactions. Ethyl lactate was chosen as the substrate because its hydrolysis is catalyzed by chymotrypsin at an easily measurable rate, it is completely miscible with water, and it is commonly available. Measurements of Kz and A$ in the simple Michaelis formulation :
0) were made over the pH range 5.2-8.6 in 0.1 M NaCl at 25°C. No buffer was used because in order to study a wide pH range, different buffers are often needed, and even with one buffer the ratios of the components change with pH. These different buffer constituents are often inhibitors or activators of enzymes and might thus mask the primary pH effect. EXPERIMENTAL Materials The or-chymotrypsin was a crystalline, salt-free product (Lot No. CD 514-17) obtained from Worthington Biochemical Co. To remove any insoluble matter from the enzyme, the Worthington chymotrypsin was dissolved in water, filtered through an ultrafine sintered-glass filter, and lyophilised. Most of the experiments 148
WCHYMOTRYPSIN-CATALYZED
HYDROLYSIS
149
were done with this preparation; however, the same results were obtained with the original material. Various sources of ethyl m-lactate were used. Most of the experiments were done with Eastman White Label grade; for some experiments the ethyl lactate was distilled through a 30-plate column at 1 cm. pressure. Preliminary experiments were made with a Mathieson, Coleman, and Bell sample. There was no difference in the kinetic behavior of these different samples. It was noted, however, that ethyl lactate once exposed to the air underwent a slow change over a period of months. Ethyl lactate which had been in a previously opened bottle for 3 months gave a lower pH when dissolved in water and hydrolyzed faster (with either base or chymotrypsin catalysis) than a fresh sample of ethyl lactate. Apparently slight hydrolysis occurred followed by reaction of the acid with the a-hydroxyl, leading to lactyl lactate and possibly higher polymers. The concentration of polyester was small because with continued hydrolysis the rate decreased and reached a value equal to that of fresh ethyl lactate. For all the experiments reported, the ethyl lactate was from recently opened bottles and gave a constant rate of hydrolysis . Reagent-grade NaCl and boiled distilled water were used in preparing all solutions.
Methods The rate of hydrolysis of ethyl lactate was measured by continuously titrating the lactic acid released. NaOH (0.1011 N) was delivered into the reaction beaker by a motor-driven Gilmont microburet. The Bodine d.c. variable speed motor was turned on or off by an amplified’ signal from a Beckman model G pH meter with external electrodes. Whenever the pH dropped below a predetermined value, base was added from the buret to keep the pH constant. The speed of the buret drive could be varied continuously from 0.033 to 45 r.p.m. by variation of the voltage to the motor, plus the use of three gears; this corresponds to a delivery rate of from 0.001 to 1.35 ml./min. with a l-ml. plunger in the buret. The pH could be kept constant to f0.05 or less with the sensitivity increasing for the intermediate rates. The volume of base added and the time were recorded manually. The reaction beaker was immersed in a constant-temperature water bath at 25.00 f .05”C. The solution in the beaker was stirred by means of a waterproofed magnetic stirrer in the bath. Nitrogen was blown over the top of the reacting solution to exclude CO2 ; in order to avoid evaporation of this solution, the nitrogen was first bubbled through a solution of approximately the same composition. The beaker was covered by a loose-fitting cap. Reaction solutions were made by mixing ethyl lactate, 1 M NaCl, and water to give a total volume of 19 ml. The rate of hydrolysis was measured for about half an hour; then 1 ml. of the enzyme solution was added, and the rate was measured for another half hour. In a few experiments the rate was measured for times up to 2 hr. The enzyme solution was made daily by dissolving the chymotrypsin in a known volume of water. The concentration was calculated from the weight of the 1 We wish to thank of the amplifier.
Professor
R. E. Powell
and Mr. H. Robinson
for the design
150
TINOCO, JR.
enzyme used and a knowledge of its water content, which was determined by drying it in vacuum at 11O’C. The concentration of enzyme in the reacting solution was 1 g./l. The hydrolysis was pseudo-zero order under the conditions used. The rate was slow so that usually only a small fraction (1%) of the ethyl lactate was hydrolyzed; the pH and the concentrations of enzyme and water were also constant, except for a slight dilution of the solutes during the reaction by the addition of the base. The total dilution was always less than 5% and often less than 1%. Rates were calculated from the slopes of plots of base volume vs. time. The plots were consistently linear. In the calculation of rate constants, the average concentrations during the reaction were used. At each pH about ten different substrate concentrations, covering the range from 0.02 to 1 M, were studied. The rate in the absence of the enzyme (which includes effects of CO, absorption) was plotted vs. substrate concentration. The appropriate rate to subtract from the rate in the presence of the enzyme could then be obtained. The rate constants k, (sec.-l X 107) for these non-enzymic hydrolyses [k, = -(dS/dt)(l/S)] were: pH 8.6, 60.2; pH 8.2, 27.2; pH 7.8, 11.3; pH 7.4, 5.92; pH 7, 3.14. For the experiments at pH’s 5.2, 6.0, and 6.5 the non-enzymic rates were obtained by linear extrapolation from the rates at higher pH’s. At the lower pH’s the non-enzymic rates were never greater than 20% of the enzyme-catalyzed rates. For one experiment a sample of diisopropyl phosphoryl (DIP) chymotrypsin was used. It was prepared by treating chymotrypsin in 0.2 M phosphate buffer at pH 7.3 with a tenfold excess of diisopropyl phosphorofluoridate (diisopropyl fluorophosphate) (DFP).* After 4 hr. at room temperature, the solution was dialyzed vs. water at 5°C. RESULTS
The kinetic parametersa ki and Ki were obtained at each pH from a least-squares treatment of Lineweaver-Burk (9) plots (Figs. l-3). From the linearity of the plots it is apparent that there is no substrate inhibition over the 50-fold concentration range studied. An exception is pH 8.6 where there is definite indication of substrate inhibition above 0.5 M. The results are given in Table I, with probable errors (10) for all values. For the calculation of ki a value of 25,000 was used for the molecular weight of chymotrypsin. There is a wide variation in the reported values of molecular weight (11-13) ; this one, based on analytical results, was chosen because hydrodynamic and light-scattering measurements are greatly affected by the well-known polymerization of chymotrypsin. In the study of a new substrate it is important to show that the * We wish to thank Dr. Irving Geschwind and the Hormone Research Laboratory of the University of California for help with this preparation. a See the Appendix for the relation between these parameters for a nn-mixture and the individual values for the D- and n-isomers.
2.5
2.0
I.5 I/V x IO' (M:'Sac.) 1.0
I
I
I
I
IO
20
30
40
I/S (M.-‘) hydrolysis of 1. Lineweaver-Burk plots for the m-chymotrypsin-catalyzed ethyl DL-lactate at pH’s 6.2, 6.0, and 6.5 in 0.10 M NaCl at 25%. The scale of the ordinate for the pH 5.2 data has been divided by ten. FIG.
.6
FIG.
2. Lineweaver-Burk of ethyl nL-lactate
plots for the a-chymotrypsin-catalyzed at pH’s 7.0, 7.4, and 7.8 in 0.10 M NaCl 151
hydrolysis at 25°C.
152
TINOCO,
JR.
I/Vx IO’ WSec.) I
I/S th4.-‘I
FIG. 3. Lineweaver-Burk of ethyl DL-lactate
plots for the a-chymotrypsin-catalyzed at pH’s 8.2 and 8.6 in 0.10 M NaCl TABLE
Kinetic
Parameters
PH
Hydrolysis
of Ethyl
in 0.10 M NaCE at WC.
.q x 10’ (sec.-l)
0.20 0.78 2.2 2.78 3.85 3.9 3.1 2.8
hydrolysis at 25°C.
I
for the a-Chymotrypsin-Catalyzed Dr.-Lactate
5.2 6.0 6.5 7.0 7.4 7.8 8.2 8.6
&--- &- -z-k-
f f f f f f f f
.04 .1 .2 .l .l .3 .5 .6
K:(M)
0.46 0.20 0.25 0.14 0.16 0.12 0.10 0.11
f f f f f f f f
0.1 .02 .03 .Ol .Ol .Ol .02 .02
apparent activity of the enzyme is not caused by other trace enzymes in the preparation. Competitive inhibition of the new activity by a specific inhibitor is good evidence for the identity of the enzyme. At pH 7.4 @phenylpropionate was studied as an inhibitor for the ethyl lactate activity. Measurements at six substrate concentrations in 0.02 M /3phenylpropionate gave a competitive inhibition constant of 0.003 M. This is in reasonable agreement with values of approximately 0.005 M
CX-CHYMOTRYPSIN-CATALYZED
153
HYDROLYSIS
obtained at pH 7.9 by Neurath et al. (14) with acetyl-L-tyrosinamide as a substrate, and thus shows that chymotrypsin is responsible for the ethyl lactate activity. It is possible, however, that some specific inhibitors of chymotrypsin would not affect this activity. For example, phenol, which is a competitive inhibitor for chymotrypsin substrates (15)) might not interfere appreciably with a chymotrypsin-ethyl lactate complex. It is also of interest to determine whether other groups on the enzyme, besides those on the catalytic site, contribute appreciably to the hydrolysis of the substrate. DIP chymotrypsin had no catalytic activity for ethyl lactate at pH 7.4. This confirms the catalysis as due to the specific esterase site of the enzyme, and differentiates ethyl lactate from the phenyl esters as substrates. Phenyl esters are hydrolyzed, though more slowly, by DIP chymotrypsin (7). This residual activity of inhibited chymotrypsin is apparently due to catalysis by the other imidazole group in the molecule (16, 17). DISCUSSION
Recent studies (8, 18, 19) have shown the need for modifying the classical mechanism for chymotrypsin action. We may write the following equation incorporating these new ideas: E+S+
ES1 -
kz
1
ES2 +p,
k,
ESs -
kh
E + Pz
(2)
‘k--a
The various enzyme-substrate complexes are: ES1 , the Michaelis complex; ES2, the serine hydroxyl-acylated complex; and ES1, the histidine imidazole-acylated complex. The products are PZ , the acid part of the substrate; and PI, the alcohol, amide, etc., portion. The symbols in Eq. (2) refer to all the ionized forms of substrates or products at a given pH, i.e., the stoichiometric concentrations. We need not consider steps involving kL2 and kL for the initial stages of the reaction when the concentrations of products are small. At a constant pH the rate of formation of products in the steady state is given by the following equations : dP1 -= dt 1 -= k;
dPz -= dt 1 1 a+k,+lc,
k%E) 1 + G/(S) 1
K~ = (k-1 + k&i 23 klkz
(3)
154
TINOCO,
JR.
The Michaelis form is retained, but the parameters have a different significance. One sees that ki is a measure of the rate-determining step, if there is one. Furthermore, Ki is in general proportional to ki. A discussion of these equations separates naturally into two parts, depending on whether kz is or is not rate determining. If kz is rate determining, we have the classical result: (Class 1)
k; = kz ;
Ki = (k-l + kZ)/kl
(6)
The step following the formation of the enzyme-substrate complex is rate determining, and Ki is an equilibrium dissociation constant if kV1 >> kz . If kz is not rate determining we have the following more complicated equations: (Class 2)
Ic4 leoa= 1 + (k-g + k.,)/ks (k--l + kz)k4 K’ = klkz[l + (k-3 + kJ/ks]
(7)
The rate is controlled by the transfer of the acyl group from the serine oxygen to the imidazole nitrogen, and its subsequent hydrolysis. pH Dependence The pH dependence of ki has often been used to determine the pK’s of groups involved in the catalytic site of the enzyme (3, 5, 8, 20) One assumes that the enzyme site is active only if it is in a certain ionized form, and that the rate of reaction of this special ionized form is pH independent. These assumptions lead to the following equations for the Michaelis-Menten formulation if one or two ionizing groups, respectively, are involved (21, 22). k: = k; =
i&o 1 + (H+>/Ko ki,o 1 + (H+)IK,
+ Ka/(H+)
For the mechanism given in Eq. (2), we must examine the pH dependence of each rate constant. There is no direct evidence about kl and k-1 ; for specific substrates where most of the binding is through
a-CHYMOTRYPSIN-CATALYZED
HYDROLYSIS
155
an aromatic ring we would expect these rate constants to be insensitive to pH, however. The pH dependence of kz is given by (8, 23):
group in where K,, 1 is the ionization constant of the imidazolium ES1 . Studies of reactions between an ester and free imidazole (16, 17) have shown that kl should have the same type of dependence:
Within the framework of the present (21,22) theories of pH dependence, one can show that Eq. (9u) requires that k-3 obey the following relation:
However, for simplicity we shall assume that k--l is pH independent in the pH range of interest. Comparing the relative stabilities of imidazolium ion (in E&) and N-acylimidazolium ion (in E&), one would expect Km,3 >> lOK,,z . This makes the pH dependence of k--) slight in the pH range where kS is important. In effect we have given the equilibrium constant (k,/kJ for the 0-acyl ti N-acyl equilibrium the type of pH dependence usually given the individual rate constants. The 0-acyl e N-acyl migrations are well known in amino alcohols (24, 25), and the pH dependence of the equilibrium agrees qualitatively with that postulated above. The rate constant kr for the hydrolysis of the acyliiidazole must have the form: k4 = k+(H+)
+ k4.w (6.0) + k4.b (OH-)
(10)
This equation usually implies that k4 will pass through a minimum in neutral solution and increase in both acid and basic solution. As no chymotrypsinor imidazole-catalyzed hydrolyses show this pH dependence, we conclude that either k4,w is very large or k4 is not rate determining. In either case we can safely take kc as pH independent. As the pH dependence of Class 1 has been described many times before (21, 22), it will not be discussed further.
156
TINOCO,
Class 2, however, is of interest. reads : (Class 2)
”
= 1 + (k-s +
kr)(l
JR.
Our pH-dependent
equation
k4 + H+/K,,z)/ks,o
now
(11)
or k4
1 + (k-3 +
k4)/ks,o
I[
1
1 + H+/Ka,z[l + ko/(k--3 +
k4)l
1(114
One sees that we have the same pH dependence given by Eqs. (8) or (9), but the ionization constant as usually determined (8, 21, 22) is an apparent one equal to K,,Jl -I- k~,~/(k-~ + k4)]. Dixon and Neurath (23) and Gutfreund and Sturtevant (8) have found that ~K,J is less than the apparent pK,,z by about 0.6 unit. Whether this change is caused mainly by the change of the ionization constant or by the effect of the three rate constants is not known. Comparing the data for ki vs. pH (Table I) with Eq. (8b), one finds values of pK, = 6.7 and pKb = 8.7 for the ethyl lactatechymotrypsin system. Whether these values refer to ES1 , or ES* and whether they are apparent values which contain [l + /~3,~/(k--3 + Ic4)] depends on the rate determining step. This question will be discussed in the next section. The prediction of the pH dependence of Ki will in general be difficult. However, as the effects of changes in IcZand k3 with pH are in opposite directions, Kg may be nearly independent of pH. It has been claimed that if h$ and Ki have a different pH behavior, then Ki is an.equilibrium constant (3, 20). This argument is clearly not valid for Class 2. Rate Determining Step Information to allow one to choose between Class 1 and Class 2 has been obtained by Gutfreund and Sturtevant (8, 18) using fast reaction techniques. Their mechanism is similar to Eq. (2) except that step 3 is absent. They have found by direct measurements that kz is not rate determining for p-nitrophenyl acetate (Class 2), and they have inferred that kz is rate determining for acetyl-L-tyrosinamide (Class 1). Dixon and Neurath (23) have obtained the same results for p-nitrophenyl acetate, and they have inferred that acetyl-L-tyrosine ethyl ester also belongs to Class 2. A comparison of the kinetic parameters obtained by various investi-
a-CHYMOTRYPSIN-CATALYZED
TABLE A Comparison
of Kinetic
Parameters Solvent
Substrate
Ethyl m-lactate Methyl hydrocinnamatt
Water
2OTo(w/w
157
HYDROLYSIS
II
for a-Chymotrypsin-Catalyzed k :,.
bec.-l)
0.04b 0.026’
LI
b
Hydrolyses
PKZ~
K:(@
6.7 7.2
0.46-0.11 (3.84.1) x 10-z
Ref.
methanol Acetyl-r-tyrosinamide
20%
(v/v)
(3)
6.7
0.045-0.18
(8)
7.3
-
(18)
2-propan01 p-Nitrophenyl
acetate
Acetyl-L-tyrosine ethyl ester Acetyl-L-tryptophan ethyl ester Acetyl-L-phenylalanine
20% (v/v) a-propan01 Water
0.028
190
6.7
Water
52
6.7
Water
150
6.85
7 x 10-t 8.9 x 10-G (1.1-3.1)
x 10-s
(5) (5) (19)
-
D Assuming a mol. wt. of 25,000 for chymotrypsin. b Maximum value. c Apparent value; see text. d The values given correspond to the lowest and highest pH’s does not pass through a maximum or minimum, these represent values.
studied. As Ki the
extreme
gators from studies of pH dependence is given in Table II. If we accept the above conclusions, p-nitrophenyl acetate and the specific amino acid ester substrates are in Class 2, while the amide substrate is in Class 1. To help place ethyl lactate and methyl hydrocinnamate we notice that the pH dependence of these two substrates passes through a definite maximum at pH 7.8, while for the last three substrates the catalytic activity is nearly constant between pH 8 and 9. While the pH dependence of h$ for acetyl-n-tyrosinamide has not been studied above pH 8, measurements at one substrate concentration show a decrease in catalytic activity above pH 8 (26). We would therefore designate the first three substrates in the table as Class 1 and the remaining ones as Class 2. That is, we think the formation of the lactylserine oxygen bond is rate determining for the ethyl lactate hydrolysis and that Ki for this system is the equilibrium dissociation constant for the Michaelis complex. The pK’s of 6.7 and 8.7 therefore refer to
158
TINOCO, JR.
ES1 ; the lower pK, must be for the active imidazolium group. As there is no known group in the chymotrypsin catalytic site with a pK = 8.7, it might be due to a more generalized pH-dependent process in the enzyme which would affect step 2 but not step 3. It is interesting that there is a large change in the optical activity of chymotrypsin at this PH (27). The variation of the pK,‘s for the different substrates is difficult to interpret because of the various solvents used and the effect of differences in i&,0/@--3 -I- k4) on the apparent pK,‘s obtained from Class 2 kinetics. The pH dependence of Kj will in general also be difficult to interpret. The magnitude of Ki for ethyl lactate, however, is of interest because it is much greater than that for other substrates. Comparing ethyl lactate with methyl hydrocinnamate, which has a similar value of ki, we see a 25- to a loo-fold difference in Ki . This difference mirrors the weaker binding between ethyl lactate and chymotrypsin. The fact that Kl for methyl hydrocinnamate is pH independent within experimental error, while Ki for ethyl lactate varies with pH, is probably caused by weak interaction of the cu-hydroxyl with some ionizable group on the enzyme. ACKNOWLEDGMENTS Dr. Susan Lowey and Mr. Donald Crothers made preliminary measurements on this system. We are very grateful for their help. We wish to thank Dr. M. P. Freeman and Professor R. J. Myers for advice on the construction of the pH-stat. We are indebted to Dr. I. I. Geschwind and Professors B. H. Mahan, H. Rapoport, and A. Streitweiser, Jr., for helpful discussion and suggestions. CONCLUSIONS
In the above discussion it has been assumed that there is a ratedetermining step; that is, that an enzyme-substrate system could be assigned to one or another class over the entire pH range. Unfortunately, even at one pH all rate constants can be of the same order of magnitude, and in general as the rate coefhcients vary with pH it is possible that different steps will beoome rate determining. However, kz and k3 have approximately the same pH dependence so that this is not very likely. We must conclude, however, that all decisions about which steps are rate limiting are tentative, except for p-nitrophenyl acetate, where the individual processes have been measured directly. That this deci-
&CHYMOTRYPSIN-CATALYZED
sion is an important one is obvious, Ki and the pH behavior depends on this information include the study of (28) and the use of product inhibition of the substrate.
HYDROLYSIS
159
as the precise interpretation of it. Other methods for obtaining different esters of the same acid by either the acid or alcohol part
SUMMARY
The pH dependence (pH 5.2-8.6) of ki and Ki for the cu-chymotrypsin-catalyzed hydrolysis of ethyl m-lactate in 0.10 M NaCl at 25°C. has been determined. The value of ki first increases with pH; reaches a maximum of 0.039 sec.-l at pH 7.8; then, decreases. The value of Ki decreases from 0.46 to 0.11 M with increasing pH. The kinetics have been discussed in terms of a mechanism involving three enzyme-substrate complexes. It has been tentatively eoncluded that the rate-determining step for ethyl lactate is the ester interchange leading to the formation of enzyme-serine lactate and the release of ethanol. A value of pK, = 6.7 was obtained for the active imidazolium group to which the lactyl is transferred before hydrolysis. APPENDIX As ethyl lactate lacks binding of substrates by point of a stereospecific L-forms of ethyl lactate
the aromatic group which is primarily chymotrypsin, and which is supposed contact with the enzyme, we would to have very similar kinetic behavior,
kZ,DI,E &I
Lz I& ;
responsible for the to provide the third expect the D- and i.e.,
K:.DL S K~.D E K~.L
However, the general relations among the various constants are:
k:.DL.= ~/G.DL.
=
&L + (K~,,/K~,D)ki,D 1 + (K~,LIK~.~)
(Al)
fb)(llK~,~,
(A21
+
~/K:,D)
The pH dependence of each step should be identical for the optical isomers according to present theory; therefore, even if k:,n is not equal to 7c:,n , the same value of pK. should be obtained. REFERENCES 1. GREEN, N. M., AND NEURATH, H., in “The Proteins” (Neurath, H., and Bailey, K., eds.), Vol. II, Pt. B, p. 1057. Academic Press Inc., New York, 1954. 2. FOSTER, R. J., AND NIEMANN, C., J. Am. Chem. Sot. 77, 1886 (1955). 3. LAIDLER, K. J., AND BARNARD, M. L., Trans. Faraday Sot. 62, 497 (1956).
160 4. 5. 6. 7. 8. 9. 10. 11.
12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.
TINOCO,
JR.
BENDER, M. L., AND KEMP, K. C., J. Am. Chem. Sot. 79, 116 (1957). CUNNINGHAM, L. W., AND BROWN, C. S., J. Biol. Chem. 221, 287 (1956). HOFSTEE, B. H. J., B&him. et Biophys. Acta 24, 211 (1957). HARTLEY, B. S., AND KILBEY, B. A., Biochem. J. 66, 288 (1954). GUTFREUND, H., AND STURTEVANT, J. M., Proc. Natl. Acad. Sci. U. S. 43, 719 (1956). LINEWEAVER, H., AND BURK, D., J. Am. Chem. Sot. 67, 658 (1934). of Physics and ChemMARGENAU, H., AND MURPHY, G. M., “The Mathematics istry,” p. 502. D. Van Nostrand, New York, 1943. GREEN, N. M., AND NEURATH, H, in “The Proteins” (Neurath, H., and Bailey, K., eds.), Vol. II, Pt. B, p. 1072. Academic Press Inc., New York, 1954. STEINER, R. F., Arch. Biochem. Biophys. 63, 457 (1954). TINOCO, I., JR., Arch. Biochcm. Biophys. 68, 367 (1957). NEURATH, H., GLADNER, J. A., AND DEMARIA, G., J. Biol. Chem. 188, 407 (1951). HUANG, H. T., AND NIEMANN, C., J. Am. Chem. Sot. ‘76, 1395 (1953). BRUICE, T. C., AND SCHMIR, G. C., J. Am. Chem. Sot. 79, 1663 (1957). BENDER, M. L., AND TURNQUIST, B. W., J. Am. Chem. Sot. 79, 1652 (1957). GUTFREUND, H., AND STURTEVANT, J. M., Biochem. J. 63, 656 (1956). DIXON, G. H., AND NEURATH, H., J. Am. Chem. Sot. 79, 4558 (1957). HAMMOND, B. R., AND GUTFREUND, H., Biochem. J. 61, 187 (1955). ALBERTY, R. A., J. Cellular Comp. Physiol. 47, 245 (1956). LAIDLER, K. J., Trans. Faraday Sot. 61, 528, 540, 550 (1955). DIXON, G. H., AND NEURATH, H., J. Biol. Chem. 226, 1049 (1957). VAN TAMELEN, E., J. Am. Chem. Sot. 73, 5773 (1951). FODOR, G., AND KISS, J., J. Chem. Sot. 1962, 1589. THOMAS, D. W., MACALLISTER, R. V., AND NIEMANN, C., J. Am. Chem. Ser. 73, 1548 (1951). RUPLEY, J. A., DREYER, W. J., ANU NEURATH, H., Biochim. et Biophys. Acta 18, 162 (1955). HOFSTEE, B. H. J., Abstracts Pacific Slope Biochemical Conference, 1957.