ACETYLCHOLINESTERASE* XII. FURTHER STUDIES OF BINDING FORCES BY IRWIN B. WILSON (From the Department of Neurology, College of Physicians and Surgeons, Columbia University, New York, New York) (Received for publication, January 16, 1952) It has been concluded in previous studies (l-3) that the active surface of acetylcholinesterasc contains two primary regions which react with substrates and inhibitors: (1) a negatively charged structure, or anionic site, which facilitates enzymic activity by attracting, binding, and orient- ing cationic substrates, and (2) an “esteratic site” responsible for the hy- drolytic activity. This site contains a basic and an acidic group. These groups must be joined by structures of high electron fluidity such as exist in conjugate double bonds. The basic group contributes to the formation of enzyme substrate and enzyme inhibitor addition compounds by forming a covalent bond with the acyl carbon atom of substrates and of inhibitors when the latter contain a carbonyl group suitably located with respect to other structural features. The acidic group does not contribute to the formation of enzyme complexes (4) but is involved only in the hydrolytic process in which it functions in conjunction with the basic group to make a combined acid-base attack upon the substrate portion of the enzyme-substrate complex, resulting in the elimination of alcohol and the formation of an acetyl enzyme (5, 6). The conclusion that the basic group reacts with the acyl carbon atom of substrates and inhibitors was reached in part from the demonstration that in a series of inhibitors of the same fundamental structure the order of inhibitory strength coincided with the order of electrophilic character of the carbon atom. It would be of interest to know whether a similar cor- respondence of elcctrophilic character and affinity exists for substrates. Such a comparison, however, camlot be made unequivocally because in no casecan the dissociation constant of an enzyme and substrate be meas- ured. What can be measured is the Michaelis-Menten constant. This constant is generally represented by -- -----~-.-. . .~ --... * This investigation wae supported by research grants from the Division of Re- search Grnnts and Fellowships of the Kational Institutes of Health, United States Public Health Service, and from the Medical Research and Development Board, Department of the Army, Office of the Surgeon General. 215 by guest on January 23, 2020 http://www.jbc.org/ Downloaded from
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ACETYLCHOLINESTERASE*
XII. FURTHER STUDIES OF BINDING FORCES
BY IRWIN B. WILSON
(From the Department of Neurology, College of Physicians and Surgeons, Columbia University, New York, New York)
(Received for publication, January 16, 1952)
It has been concluded in previous studies (l-3) that the active surface of acetylcholinesterasc contains two primary regions which react with substrates and inhibitors: (1) a negatively charged structure, or anionic site, which facilitates enzymic activity by attracting, binding, and orient- ing cationic substrates, and (2) an “esteratic site” responsible for the hy- drolytic activity. This site contains a basic and an acidic group. These groups must be joined by structures of high electron fluidity such as exist in conjugate double bonds. The basic group contributes to the formation of enzyme substrate and enzyme inhibitor addition compounds by forming a covalent bond with the acyl carbon atom of substrates and of inhibitors when the latter contain a carbonyl group suitably located with respect to other structural features. The acidic group does not contribute to the formation of enzyme complexes (4) but is involved only in the hydrolytic process in which it functions in conjunction with the basic group to make a combined acid-base attack upon the substrate portion of the enzyme-substrate complex, resulting in the elimination of alcohol and the formation of an acetyl enzyme (5, 6).
The conclusion that the basic group reacts with the acyl carbon atom of substrates and inhibitors was reached in part from the demonstration that in a series of inhibitors of the same fundamental structure the order of inhibitory strength coincided with the order of electrophilic character of the carbon atom. It would be of interest to know whether a similar cor- respondence of elcctrophilic character and affinity exists for substrates. Such a comparison, however, camlot be made unequivocally because in no case can the dissociation constant of an enzyme and substrate be meas- ured. What can be measured is the Michaelis-Menten constant. This constant is generally represented by
-- -----~-.-. . .~ --... * This investigation wae supported by research grants from the Division of Re-
search Grnnts and Fellowships of the Kational Institutes of Health, United States Public Health Service, and from the Medical Research and Development Board, Department of the Army, Office of the Surgeon General.
This representation is, however, seldom, if ever, correct, since it is based upon a model which does not t,ake into account the dissociation of acidic and basic groups upon which the activity depends. Only certain pH- dependent forms yield active complexes. For acetylcholinesterase (and a non-dissociating substrate) the constant is given by
K = kr + kp 1 + (H+)
[ -
Km n
h K BH2+ +(H+) 1 where (Hf) is the hydrogen ion activity, KRH2+ is t.he acid dissociation constant of the basic group, and KEH (5 X 10W”) is the dissociation con- stant of the acid group of the enzyme-substrate complex. For pH less than 9 the term containing Ksa is negligible but the term containing KxH2+ may be quite large.
The value of KBHp + is uncertain, since several different kinds of evalua- tions, sueh as from the pH dependence of acetylcholine and thiolacetic acid hydrolysis, and of prostigmine inhibition, have yielded values ranging from 0.7 X lo-’ to 3 X lo-‘. The best value appears to be 1 X lo-‘; hence at pH 7.3 the bracketed term equals 1.5. In comparing the con- stants of different substrates the value of this term is of no importance if measurements are made at the same pH.
In general it is possible to measure only K, and ka; thus the enzyme- substrate dissociation constant kJkl is unknown. In the case in which kz is larger than k, the dissociation constant is approximated by K,. It will be seen that there is some evidence that this condition prevails for acetylcholinesterase and the substrates investigated in this study.
This report is also concerned with an analysis of the r61e of methyl or other alkyl groups in the interact.ion of smaller molecules with the enzyme.
Methods
Kinetic measurements were made manometrically (7) for all inhibition experiments and for the substrates ethyl acetate, ethyl chloroacetate, isoamyl acetate, and dimethylaminoethyl acetate. In those cases in which the inhibitor or substrate can exist in acidic and basic forms, some of the acid produced during hydrolysis is utilized in shifting this equilib- rium and does not go into the production of carbon dioxide. The magni- tude of this effect depends upon the equilibrium constant and the con- centration and was negligibly small in all cases in which the concentrations were 0.1 M or less, except for ammonium chloride, dimethylaminoethyl
acetate hydrochloride, and ethanolamine hydrochloride. The corrections for this effect were determined experimentally by adding a known amount of acid from the side arms in the absence of enzyme. The decrease of CO* with ammonium chloride is so large at the very high concentrations of 2 M necessary for inhibition that this method cannot yield satisfactory results. In any event, the salt concentration is so high that it would be difficult, if not impossible, to assign significance to any observed inhibi- tion. Therefore, data are not included for ammonium chloride.
Even at the highest concentrations the corrections for ethanolamine hydrochloride and dimethylaminoethyl acetate hydrochloride were suf- ficiently small so that they could be applied with accuracy. The latter compound has an additional correction due t>o the fact that at pH 7.3 only 90 per cent of the substrate molecules are cationic, whereas nearly 100 per cent of the dimethylethanolamine produced on hydrolysis is cationic. There will thus be a 10 per cent retention of carbon dioxide.
In all instances measurements were made over a range of concentrations, but in the case of cationic inhibitors the ionic strength was held constant in any one series by balancing the decrease in inhibitor with an increase in sodium chloride in those cases in which the amount of inhibitor was significant with respect to the usual 0.25 M ionic strength of the bicar- bonate buffer.
In the case of acetic anhydride the calorimetric method of assay was used (8). 1 mole of acetic anhydride yields 1 mole of acetohydroxamic acid. Readings were taken within a few seconds of adding substrate and half a minute later. Corrections for non-enzymic hydrolysis, while quite high, were less than 20 per cent; hence reasonably accurate results could be obtained.
The rates of hydrolysis of acetylcholine and dithiolacetic acid were measured by iodometric assay of the mercaptan group so produced. The solutions of dithiolacetic acid were obtained by adding the stoichiometric amount of iodine to thiolacetic acid solutions. There is a slow spon- taneous hydrolysis of this compound. The reaction is quite interesting in that there is a precipitation of sulfur. The reaction appears to be
CHaCOSSCOCHa + H& + CIIaCOOH + CHsCOSSH
C&OSH + S
In the case of acetylthiocholine the concentrations were so low that it was necessary to use volumes up to '75 ml. to obtain satisfactory accuracy. The reactions mere carriCd out in phosphate buffer of the same magnesium concentration and ionic strength as the manomctric medium. The re- actions were stopped by adding a high concentration of prostigmine.
In all cases the enzyme was purified cholinesterase from the electric organ of Electrophorus electricus (9).
Results
The inhibitory strength of various ammonium ions is recorded in Table I in terms of the concentration required to produce 50 per cent inhibi- tion (CM) at pH 7.3 and an acetylcholine concentration of 4 PM per ml.
The inhibition caused by these ions is reversible and competitive; hence inhibition is dependent upon substrate concentration and is greater at
TABLE I Inhibitory Strength oJ Various Ammonium Ions
C&O = 50 per cent inhibition concentrations. _.----
Tetramethylammonium ion ................ Trimethylammonium ion ..................
“ “ ................... Dimcthylammonium ion .................. Metl~ylammoniumion ..................... Trimethylethanolammonium ion (choline). Dimethylethanolammonium “ ........... Methylethanolammonium ion .............. Ethanolammonium ion .................... Isoamyl alcohol. .......................... Tetraethylammonium ion .................. Dimethylpropylammonium ion ............. Trimethylphenylammonium “ ............ IIexyldimethylaminoethanolammonium ion
. .
1.8 X lo-’ 1.5 x lo-’ 2.1 x 10-p* 1.2 x lo-‘* 7.0 x lo-**? 5.0 x 10-3 5.0 x 10-s 7.0 x 10-2 2.8 X lo-‘+t 1.5 x 10-1 5.0 x 10-S 4.0 x 10 a 8.0 X lo-’ 4.5 x 10-s
* Ionic strength 0.45 ar; others about 0.25 M. t Extrapolated from a concentration of 0.20 M.
lower concentrations. Thus, while choline at a concentration of 0.005 M inhibits 50 per cent when the acetylcholine concentration is 4 C.~M per ml., it inhibits 60 per cent when the substrate concentration is 2 PM per ml., and only 35 per cent when it is 8 pM per ml.
The CW values were obtained by measuring the activity at different con- centrations of inhibitor and plotting the data so as to obtain a linear locus of points in accordance wit.h the equation
tP --lf- (I) 0 K
I 1 + (s) + Wf --I Km KJG
where v” is the rate without inhibitor, v with mhibitor, (I) the inhibitor
concentration, Kr the inhibitor enzyme dissociation constant, K&032 the dissociation constant for the inactive enzyme-substrate super com- plex E&., and (S) the concentration of substrate. This equation indicates
TABLE II Values of &, k,, and Concentration Range for Several thbulrales
5 x 10-L3 x 10-I 5 x 109-e x 10-a 6 X lo-‘-5 X 10-J 1 x 10-2-1 x IO-1
. 4 x lo-cl x lo-’ 5 x 10-q x 10-J 3 x 10-c3 x lo-’ 2 x lo-cl x 10-l
6 x lo-’ 12 3 x lo-* 13 8 x lo-’ 11 1 x 10-J 38 4.5 x lo-’ 100 4 x lO-’ 13 2 x lo-’ 18 1.2 x lo-’ 100
Rekive kr
* Taken from Wilson and Bergmann (2).
100
90
FIG. 1. (a) Dimethylaminoethyl acetate hydrochloride, (b) acetylcholine chlor- ide, taken from Auguetineson and Nachmansohn (ll), (c) acetylthiocholine iodide. The optimum velocity of acetylcholine hydrolysis was given a reference value of loo.
that the enzyme inhibitor dissociation constant is equal to 0.09 X CW for our conditions.
The values of K, and ka were determined in the usual way (10) from a
linear plot of the reciprocal of the reaction velocity against the reciprocal of the substrate concentration. This method involves an extrapolation to infinite substrate concentration and is therefore not apt to yield precise values in those cases such as ethyl acetate, isoamyl acetat.e, and ethyl ch1oroacetat.e in which the substrate concentration is limited by solubility and the highest reaction velocity measured is only about one-half the calculated maximum. However, since satisfactory straight lines were obt.ained, it seems unlikely that the error is greater than 50 per cent.
Values of K,, ka, and the concentration range in which measurements were made are present4 for several substrates in Table II. Fig. 1 shows a comparison of the velocity-substrate relationship of acetylthiocholine (iodide) and dimethylaminoethyl acetate (hydrochloride) with acetyl- choline (chloride). The acetylcholine data are taken from August&son and Kachmansohn (11). The acctylthiocholine curve is not quite sym- metrical, and so the data do not closely follow the usual formulation of a bell-shaped curve based on a formula of ES2 for the inactive super com- plex but t.he fit is close enough to yield satisfactory values of ka and K,.
DISCUSSION
Calionic Inhibiifors-The fact that the successive removal of methyl groups starting with the trialkyl member of the simple ammonium ion and ethanolammonium ion series leads to progressively poorer inhibitors reveals the importance of van der Waals’ dispersion forces in the binding of these ions to the enzyme. On the average, there is a 6.7-fold diminu- tion of inhibition for each methyl group corresponding to some 1.2 kilo- calories per mole change in the free energy of binding. This quantity can easily be accounted for on the basis of dispersion forces between the sub- strate and a hydrocarbon moiety of the enzyme. These forces, for ex- ample, are the only ones of significance operating between methane mole- cules and are responsible for the liquefaction of that gas. The internal energy’ of evaporation (at the boiling point) of methane is 2.0 kilocalories per mole (12); therefore there is ample energy available from this effect.
That t.hc addition of a methyl group to the trialkyl cation of bot,h the ammonium and ethanolammonium series produces little or no change in the binding indicates that those ions do not cause a folding of the protein. These ions are more or less spherical; hence it is possible for a fourth alkyl group to come into close proximity to the enzyme only if t,he protein en- gulfs the ion. Otherwise, and this seems to be the case, the fourth group will project away from the enzyme into the surrounding aqueous medium and will be without binding properties.
Increasing the size of the alkyl groups compared to tetramethylam-
monium ion, as in tetraethylammonium ion, choline or dimethylamino- ethanolammonium, dimethylaminopropane ammonium ion, and hexyldi- methylaminoethanolammonium ion produces increased binding, and again this effect is readily attributed to van der Waals’ forces. Trimethyl- phenylammonium ion is rather better than the other cations, but it is not clear whether this is due to some special effect of the aromatic ring or just increased size. Increasing the size of alkyl groups cannot, of course, in- definitely lead to greater binding, since soon a bulk may be reached which prevents a very close approach of enzyme and inhibitor.
Dimethylaminoethanolammonium ion is about 30 times as effective as its uncharged analobqe isoamyl alcohol. This increase is a simple elec- trostatic interaction of the cation with the anionic site. The cation of eserine is about 20 times more powerful than the uncharged molecules and this difference corresponded to an approach of the centers of electric charge to within 6.5 A (1). The present data indicate an approach to about 5.7 A as compared to the closest approach of 5 A estimated from bond radii, if 3.5 A is taken for the radius of the methylated ammonium group and 1.5 A for the unknown negative group in the anionic site.
Trimethylammonium ion decreases in inhibitory power as the ionic strength is increased. Presumably the other cations would show the same de&ease, since this observation is in agreement with the decrease observed by Myers (13) with prostigmine and cserinc. We do not, however, agree with his interpretation that sodium ions compete with prostigmine for the anionic site. There is no indication that the binding energy would be anywhere near sufficient to produce significant binding, and, moreover, it is not clear how these ions can compete with prostigmine and not simul- taneously wit.h acetylcholine when the concentrations of the substrate and inhibitor are such that they mutually compete. The salt effect may involve the effect of ionic strength on act,ivity coefficients, i.e. the Br$nsted primary salt effect.
The ammonium ions inhibit by occupying the anionic site. The bind- ing is effectuated by ionic and dispersion forces. In the case of uncharged analogues such as isoamyl alcohol, isoamyl acetate, and the uncharged forms of dimethylaminoethyl acetate and eserine, only the van der Waals’ forces arc involved in binding at this site. By occupying the anionic site these inhibitors not only make the site unavailable for binding a substrate molecule but sterically interfere wit,h binding at the esteratic site or with the conversion of the complex to the critical complex. These compounds, for example, can completely inhibit the enzymic hydrolysis of ethyl ace- tate. But, if both the substrate and the inhibitor are sufficiently small, steric inhibition will not be complete and complete inhibition cannot be achieved. The inhibition of thiolacetic acid hydrolysis by trimethyl-
ammonium ion, for example, reaches a saturation value of only 30 per cent (6).
The fact that choline is no better than dimethylaminopropanc hydro- chloride as an inhibitor indicates that no hydrogen bond is involved in the binding of choline and suggests that no hydrogen bond is involved in binding acetylcholine. This is in agreement with the finding that the acidic group of the esteratic site does not form a hydrogen bond with prostigmine (4), although it is believed that a kind of hydrogen bond is involved in the critical complex formed with substrates and helps explain thereby the low energy of activation.
Substrates-The substrates listed in Table II have K, values decreasing from top to bottom by a factor of 3 X 103, whereas the lig values differ by a factor of 9. In the series ethyl acetate, ethyl chloroacetate, isoamyl acetate, and acetic anhydride, K, changes a thousand fold, without sig- nificant change in ka. Acetylcholine and acetylthiocholine have the same value of k3, but K, differs by a factor of 3.7.
From this lack of relationship between kS and K,, it is tempting to assume that K, is determined by kJkl; i.e., that K,,, (corrected for the PI-I-dependent term of the equation given above) is the enzyme-substrate dissociation constant, but it is still possible that ko/kl is a comparable or even larger term. However, in that event a negative change in K, would necessarily mean a positive change in kl; yet the addition of methyl groups in going from ethyl acetate to isoamyl acetate would hardly be expected to increase kl. Moreover, if an increase in 121 were not in turn to indicate a decrease in kz/kl, it would be necessary for kz to increase correspondingly. But just those structural features, which the inhibition experiments have shown to produce greater binding and would therefore be expected to reduce kg, do in fact decrease K,. We can, for example, compare ethyl acetate, isoamyl acetat,e, and acetylcholine. From Table I we see that two methyl groups increase the binding by about a factor of 45 and a positive charge by a factor of about 30; therefore, the binding of acetyl- choline should be about 1300 times greater than ethyl acetate. The K, value is smaller by about a factor of 1100. Similarly, isoamyl acetate should be about 45 times better bound than ethyl acetate. Its K, is smaller by a factor of 60. However, on t.he same basis we would expect dimethylaminoethyl acetate to have the same K, as acetylcholine, but its value is larger by a factor of 2.2. From these considerations it seems quite safe to relate large changes in K, with changes in binding. Sub- stituting a sulfur for an oxygen atom in an organic compound has an effect which can be readily explained on the basis of increasing the electrophilic character of neighboring carbon atoms. This is illustrated, for example, by the greater acidity of thio ethers, thiol acids, and mercaptans relative
to the corresponding oxygen compounds. Even thioglycolic acid is stronger than glycolic acid. Moreover, thiolacetic acid is an acetylating agent (14). Hammett (15) has assigned a greater u value to CH&- than to CH#--. With this in mind we can arrange the substrates in two series of increasing elect.rophilic character of the acyl carbon atom as (1) ethyl acetate, ethyl chloroacetate, acetic anhydride, dithiolacetic acid, and (2) acetylcholine, acetylthiocholine. This is the order in which the K, de- creases. Within the uncertainty discussed above, it is, t.herefore, the order in which binding increases, and so the importance of an electrophilic acyl carbon atom in forming enzyme complexes is further substantiated. The clectrophilic character appears to be without effect on ka. The satu- ration rate of such an easily hydrolyzed substance as acetic anhydride is no greater than that of ethyl acetate. That the ks values are different for different substrates shows that the rate-cont,rolling step cannot be t.he reaction of acetylatcd enzyme wit.h wat,er which would be identical for all of the substrates except the chloroacetate and must, therefore, be the rate of formation of acctylated enzyme. Actually, this argument can apply strictly only to all but the fast& reaction; in the case of acetylcholine and acet,ylthiocholine we cannot be certain that the rate-controlling step has not shifted to the hydrolysis of acetylated enzyme.
dcetic anhydride and dithiolacetic acid have about the same K, as acetylcholine, although they lack the methylated cationic structure which is so readily bound by this enzyme. Evidently this lack is amply com- pensated by the highly electrophilic acyl carbon atoms.
The acetylthiocholine velocity-substrate concentration curve is very nearly the same as that of acetylcholine (Fig. l), but displaced to about one-f0urt.h of the concentration; hence this substrate is more readily hy- drolyzed at very low concentrations than is acetylcholine.
As already mentioned, judging from the above inhibitors, we should have expected that dimethylaminocthyl acet.ate would be bound by the enzyme in a nearly identical manner as acetylcholine. Actually, the K, and ka values differ by a small but significant factor of about 2.5. The inhibitors referred to above are bound only at the anionic site. It would appear that when we have binding at both the est.eratic and anionic sites the “cxt.ra” methyl group is not inert. Yet for this methyl group to come into close proximity with the enzyme would appear t.o require a reshaping of the protein, as discussed above, wit,h regard to the inhibitors. Such a change might, be of quite considerable inter&. The differences in ka and K, are very slight when compared to the remarkable difference in the biological activity of the two compounds. There the difference is a hun- dred fold or more (16). The choline acetylating system acet.ylates di- methylaminoethanol just as well as choline (17). But from Fig. 1 we
again see a distinction between the two compounds: dimethylaminoethyl acetate does not produce a bell-shaped curve and it does not show sub- strate inhibition. The “extra” methyl group is somehow involved in the formation of t.he inactive super complex. It would, therefore, seem likely that the super complex involves some sort of reshaping of the protein molecule. Nachmansohn was led to postulate that during the propaga- tion of nerve impulses acetylcholine is released, reacts with a receptor pro- tein, and thereby changes the permeability of the protein membrane (18). The possible reshaping ability of acetylcholine in the case of the enzyme protein is very interesting, especially in view of the lack of this ability in the biologically inact,ive dimethylaminoethyl acetate. The concentrations of acetylcholine which affect the enzyme arc, of course, from the biological point of view extremely high, and hence the enzyme cannot be the re- ceptor protein.
SUMMARY
Inhibition studies with alkylated ammonium ions reveal that the alkyl group has binding properties for acetylcholinesterase whose magnitude is readily accounted for on the basis of dispersion forces. The tetraalkyl and trialkyl ions are equal inhibitors, which might be expected since these ions are spherical and a close proximity of t,he fourth group with the en- zyme would require that the protein engulf the molecule; i.e., a reshaping of the protein would be required. The importance of a positive electrical charge is confirmed.
Studies with different substrates show that increases in binding are reflected in the value of K,, whereas kz bears no relationship to K,,,. The importance of an electrophilic carbomatom is shown. The rate-control- ling step in the hydrolysis of substrates is the formation of acylated en- zyme. The substrates acetylcholine and dimethylaminoethyl acetate have K, and k3 values which differ only by a factor somewhat greater than 2; then interaction with the enzyme is not very different at low con- centrations, but at high concentrations there is a marked difference in that the dimethyl compound does not show substrate inhibition. This dif- ference is discussed in relationship to a possible reshaping of the protein and its relationship to the marked difference in biological activity of the two compounds.
The rate-substrate concentration curve for acetylthiocholine is similar to that of acetylcholine but displaced to one-quarter of the concentration.
The author wishes to express his grat.itude to Dr. D. Kachmansohn for his inspiring guidance and advice throughout this work. He also wishes to thank Mrs. Ida Freiberger for her assistance in performing the experiments.
1. Wilson, I. B., and Bergmann, F., J. Biol. Chem., 186,479 (1950). 2. Wilson, I. B., and Bergmann, F., J. Biol. Chem., 166, 683 (1950). 3. Bergmann, F., Wilson, I. B., and Nachmansohn, D., J. Biol. Chem., 166, 693
(1950). 4. Wilson, I. B., B&him. et biophys. acla, ‘7, 466 (1951). 5. Wilson, I. B., Bergmann, F., and Xachmansohn, D., J. Biol. Chem., 186, 781
(1950). 6. Wi$son, I. B., Biochim. et biophys. ada, 7, 520 (1951). 7. Sachmansohn, D., and Rothenberg, hl. A., J. Biol. Chem., 168. 653 (1945). 8. Ilestrin, S., J. Biol. Chem., 180, 249 (1949). 9. Rothenberg, M. A., and Kachmansohn, D., J. Biol. Chem., 166, 223 (19.47).
10. Lineweaver, II., and Burk, D., J. Am. Chem. Sot., 58, 658 (1934). 11. Augustinsson, K.-B., and Nachmansohn, D., J. Biol. Chem., 1’79, 543 (1949). 12. International critical tables of numerical data, physics, chemistry and tech-
nology, New York, 6 (1929). 13. Myers, D. K., Arch. Biochem., 2’7, 3-11 (1950). 14. Pawlewski, B., Her. them. Ges., 31, 611 (1898). 15. Hammett, L. P., Physical organic chemistry, New York (1940). 16. Stehle, R. L., Melville, K. J., and Oldham, F. K., J. Pharmacol. and Exp. Therap.,
66. 473 (1936). 17. Korey, S. R., de Braganza, B., and Kachmansohn, D., J. Biol. Chem., 169, 705
(1951). 18. n’achmansohn, D., in Barron, E. S. G., Modern trends in physiology and bio-
chemistry. New York, 229 (1952). by guest on January 23, 2020http://w
