Nov. 5 , 1958
COMPETITIVE INHIBITION OF ACETYLCHOLINESTERASE SURFACE
acetate. The organic extract was dried over NazSO4 and evaporated to near dryness. The residual acetic acid was removed in vacuo over NaOH. The residue was recrystallized from ethyl acetatebenzene and sublimed a t 240' and 0.01 mm. The product melted a t 300-3051' (evacuated (c = 0.5, diox.), Xg:io" 2% mp capillary), [ O ~ ] ~+310° 'D (3,600). Repeated recrystallization from methanol gave analytically pure material containing one mole of methanol of crystallization. Anal. Calcd. for ClaHnOa.CH30H: C, 71.67; H, 8.23. Found: C, 71.55; H, 8.23. 1,4-Diacetoxy-l,3,5 (IO)-estratriene-17-one (XVI) .-The crude hydroquinone XV from reduction of 60 mg. of quinone XIV was dissolved in 1.0 ml. of pyridine and treated with 0.20 ml. of acetic anhydride. After standing on the steambath 20 min. the solution was treated with 5 drops of water and allowed to stand a t room temperature for 10 min. The reaction mixture was then worked up in the usual way. Sublimation and repeated crystallization from cyclohexanebenzene gave a product that melted a t 163.0-163.6", [ C Y ] ~ ~+273" D (c 1.0, CHCla), 265 mp (340).
5687
Anal. Calcd. for CZ~H2606: C, 71.33; H, 7.07. Found: C, 71.36; H , 7.06. 6-Dehydroestrone (XXI) .--.4solution of quinol I1 (60 mg.) in 10 ml. of methylene chloride was treated with 0.20 ml. of PBr3 and allowed to stand a t room temperature for 16 hours. The solution was shaken with 10 ml. of water for 10 minutes and the organic phase was extracted with 10 ml. of 10% KHCOa solution. The combined aqueous extracts were acidified with concd. HC1 and heated on the steam-bath for one hour. Extraction of the cooled solution with chloroform produced a quantity of crystalline solid contaminated with a purple pigment. Recrystallization followed by sublimation in vacuum and recrystallization from methanol gave 5 mg. of colorless crystals, m.p. 259261.5' (evacuated capillary). The identity of the product was verified by comparison of infrared and ultraviolet spectra with those of authentic 6-dehydroestr~ne.'~A trace of impurity having X ~ ~ : o a 291 mp appeared to be present. The melting point was not depressed by admixture of 6dehydroestrone. SHREWSBURY, MASS.
[CONTRIBUTION FROM THE DEPARTMENT O F CHEMISTRY, GEORGETOWN UNIVERSITY, THE NATIONAL INSTITUTE FOR ARTHRITIS AND METABOLIC DISEASES,AND THE NAVAL MEDICAL RESEARCH INSTITUTE]
The Acetylcholinesterase Surface. IX. Dependence of Competitive Inhibition by Diaminocyclohexane Derivatives on Substrate Level'" BY D. S. MASTERS ON,'^ S. L. FRIESS AND B. WITKOP RECEIVEDAPRIL16, 1958 Certain features of the inhibition of the system acetylcholinesteraseacetylcholine by the cis and trans isomers of 2dirnethylaminocyclohexyltrimethylammonium iodide (111 and IV) have been shown to depend markedly on the relative substrate levels employed, At pH 7.4 and 25' with a protein concentration of the order of 2 X lo-' mg. per ml., inhibition M and lower, but is found to deviate by each of these isomers is apparently competitive a t substrate levels of 1.5 X from the competitive relation a t higher levels. A possible model to account for this behavior as well as the feature of inhibition of the system by excess substrate has been discussed. The amide V in the trans series of diamine derivatives has been found to be inert to the catalytic action of the enzyme in hydrolysis reaction.
Previous kinetic studies of the system acetylcholinesterase-acetylcholine (AChE-AC) have pointed t o the competitive nature of the inhibition process characteristic of such reversible inhibitors as eserine2 and certain substituted ethylenediamines.a However, for the tertiary and quaternary compounds I and I1 reversible inhibition in the
Q (
CI-
Me
CICHGH21!JL(CH2!fi-) I
Me2N OH (cix) I
rations of diaminesb and aminoalcohols and acetatesjBmade it a matter of considerable interest to investigate the inhibitory properties of the cis- and trans-diamine derivatives I11 and I V , particularly
I
2
Me
I1
substrate concentration range up t o 3 X M was found to be clearly non-cornpetiti~e,~ with the intrinsic inhibitory power of either compound a t pH 7.4 independent of the initial acetylcholine concentration employed. These observations and their implications with respect to surface equilibria, coupled with the marked ability of the surface to distinguish between stereochemical configu(1) (a) The opinions in this paper are those of the authors and do not (b) Taken necessarily reflect the views of the Navy Department. in part from the M.S. thesis of D. S. Masterson, Georgetown University, 1957. (2) K. B. Augustinsson and D. Kachmansohn, J . B i d . Chem., 179, 543 (1949). (3) (a) S.L. Friess and W. J. McCarville, THISJ O I J R X A L , 76, 1363 (1954); (b) S. L. Friess and H. D. Baldridge, ibid., 7 8 , 199, 966 (1956). (41 S. L. Friess, ibid., 79, 3269 (1957).
Mesh+ NMe2 111
Me3N+ IV
1
MeaNi
v
in regard t o the competitive or non-competitive character of their inhibition. These comDounds possess both the quaternary ammonium finction and the center of high electron density, with appropriate separation distances between the two, that might be expected3a to lead to significant inhibitory activity in the AChE-AC system. This study has been carried out a t several substrate concentrations on the low branch of the [substratelo vs. activity profile, and has been supplemented briefly by enzymatic hydrolysis experiments with the closely related amide V. Results Inhibition data from kinetic experiments involving 111and IV were fitted to linear V / V I ers. [I]plots ( 5 ) S. L. Friess, E. R. Whitcomb, Bart T.Hogan and P. A. French, Arch. Biochein. and Biophys., 74, 451 (1958). (6) H. D. Raldridge, W.J. hlccarville and S. L. Friess, THISJOURNAL, 77, 739 (1955).
D. S. MASTERSON, S. L. FRIESS AND B. WITKOP
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Vol. 80
to 1.5 X M the inhibition by each diamine derivative appears to be competitive in nature, since the operational K I (comp.) values calculated by equation 1 for each compound are sensibly constant with changing [substrate] levels, while the K I (non-comp.) values derived from (2) steadily rise with varying substrate. However, when [subM level this aps t r a t e ] ~is raised to the 3 X K m [I1 "JI = 1 ( 1) parent competitive behavior breaks down, as indiKI(Km [Slo) cated by a dropping of K I (comp.) values from Z'/VI = 1 ( [I I/&) (2) their previously constant levels and the continued where v = uninhibited velocity and VI = inhibited rise of K I (non-comp.) values. However, alvelocity. Each observed slope was used to calcu- though neither law appears to be obeyed uniquely late the corresponding K I values a t the given sub- near this substrate level, the v / v ~vs. [I] plots are strate level, according to 1 and 2. The value of Km still quite linear with intercepts of unity, as rerequired in (1) was experimentally determined to be quired by both equations 1 and 2. (2.23 f: 0.02) x under the present conditions, This behavior pattern covering the low concenfrom four series of kinetic runs over the iSC range 2 - tration branch of the bell-shaped activity vs. [SI 10 X M and the application of the familiar profile" for the AChE-AC system can be rationLineweaver-Burk equation* for a system obeying alized in terms of a t least one general scheme (all Michaelis-Menten kinetics. K's are association constants). The values of K I calculated for competitive and Ki K. non-competitive inhibition by the diamine derivaE S ES S ESn (inactive) tives I11 and IV are summarized in Table I.
by the method of least squares, for the calculation v i a observed slopes of K I values for dissociation of the enzyme-inhibitor complexes. Equations 1 and 2 for competitive and non-competitive inhibition,' respectively, are linear in v / v ~vs. [I], but (1) furnishes a slope dependent on [SI0 whereas the slope in (2) does not contain substrate dependence.
+
+
INHIBITIONOF CornScries
1 2 3 4 5 6"
pound
I11 I11 I11
I11 I11 I11 IV 8 IV I\' 9 IV 10 IV 11 a This series tion volume of
-
+
TABLE I XChE-AC BY COMPOUSDS 111 AND I V 25.00 =t0.05" ASD pH 7.40 [Substrate] 0
Competitive K I X 105
M X 10s
+I
e
+
+I
I
kz
L--+
E
-+ products
AT
K'i
Non-competitive K~ x 104
1.88 f 0.02 1.00 3.42 i 0.02 1.50 3 . 6 0 i .07 j 2 . 7 9 f .05 1.50 3.60 & .05 12.79 f .04 3.00 2.44 & .03 3.53 f: .04 3.40 f .05 3.00 2.35 i. .03 3.36 2.06 f: .08 2.03 f .03 1.00 3.69 i .05 2.89 zk .03 3.73 i .04 1.50 3.02 f .02 3.90 i .02 1.50 4 . 0 6 + .03 2.80 i .02 3.00 4.48 + .03 3.00 3.09 i .02 was carried out at 25.14 + 0.03" in a reac6.40 ml. at pH 7.4.
1
I 1
Several interesting points emerge from the data of Table I. First, on the basis of either the competitive or non-competitive strength index a t each substrate level, the cis compound I11 is a slightly more potent inhibitor (with smaller K I value) than the trans derivative IV. This is in accord with on, ~ the cyclic 1,2-aminoprevious o b s e r ~ a t i o n ~ alcohols and acetates, in which the small separation distance between the two polar functions of cis derivatives appears to offer a better fit to the bifunctionallo catalytic unit on the enzymatic surface than that in the corresponding trans compounds with their larger inter-group spacings, and is also in line with the generally accepted picture of binding of inhibitors or substrates by twopointed interaction with the AChE surface. More striking than this, however, is the observation that in the substrate concentration range up (7) See P. W , Wilsoti i n "Respiratory Enzymes," H . A%. Lardy, ed., I3urgess. hIinne:rpolis, M i n n , 1%4
