II
Mary Ann Carper and Roberl W. Carper Wichita State University Wichito, Konsos 67208
Biochemistry: tholinesterase
During the past five years we have tried several enzyme systems in an attempt to find one with a number of different variables that would be appropriate for an undergraduate biochemistry laboratory. Moreover, we are interested in experiments in which the class may he split up into groups of between two and four students such that each group may perform separate tasks. The results of each group are then integrated to form sets of data and the class discusses and analyzes the data. We have found that the students enjoy this approach and are quite critical of one another, thus relieving the instructor of certain psychological burdens which plague all of us. After various systems were tried, we finally came to one of the cholinesterases and this proved to be ideal in a number of respects. In particular, cholinesterases are enzymes that possess the ability to hydrolyze various esters of choline to the appropriate alcohol and acid (1)
-11
n
(c~,),ih~~.oco~ + H,O
E ~t
nitrogen while the latter accomplishes the actual hydrolysis. On the other hand, the non-esteratic (anionic) site of BuChE is less influenced by charge and more concerned with steric and other effects than is the anionic site of AChE (13, 19-22). The following mechanism for the esterases is now generally accepted EH
+
AB
k,
EH-AB
EA
+
BH
(2)
k-1
EA
+
k
H,O A M
+
AOH
(3)
where AB = ester, BH = alcohol or H z 0 (ACh), and AOH = acid. In cases where inhibition may be effected by various esters such as carbamates and organophosphates, the following overall reaction dominates (14, 16)
+
GHJ,NC,H,OH + RCOOH (1) The most famous of the cholinesterases is acetylcholinesterase (acetyl hydrolase, EC 3.1.1.7) which is often referred to as the "true cholinesterase." This particular designation results from the fact that butyrylcholinesterase ( I ) (EC 3.1.1.8) (BuChE) catalyzes acetylcholine and hutyrylcholine reactions while acetylcholinesterase (AChE) is incapable of utilizing hutyrylcholine as a substrate (2). Current theory (3, 4) maintains that the action of acetylcholine (ACh) initiates certain reactions which are responsible for the increased ion permeability of membranes during electrical activity. The level of AChE will then control the hydrolysis of acetylcholine and consequently the rate of ion flow through the membranes. In particular, Nachmansohn (3) has postulated that ACh is released by excitation in the excitable membranes as a specific signal and causes a conformation change of the ACh-receptor, thereby releasing CaZ+ ions which are hound to the protein. The released CaZ+ ions may induce conformation changes of phospholipids and other polyelectrolytes. The net result is a change of ion permeability and the consequent flow of large numbers of ions across the membrane per molecule of ACh released. However, AChE can hydrolyze ACh, thus permitting the receptor protein to return to its original conformation and thus reestablish the previous ion harrier. The above hypothesis has been supported by work on axonal membranes, etc. (5-7), and recently, Changeux et al. (8) and Miledi et al. (9) have reported the isolation of the receptor protein as located in the membranes of eel electric tissue. Furthermore, these reports (8, 9) indicate that the cholinesterase active sites and the receptor sites are equal in number. This major breakthrough strengthens the previous theory (3, 4) and suggests that other important discoveries are just over the horizon. Studies of the active sites of AC and BChE have yielded a substantial amount of information about their individual characteristics. Typical (10-17) studies utilize various substrates and inhibitors to ascertain the types of forces that are functioned a t or near the active site. In particular, i t appears that AChE possesses both an anionic and an esteratic site. The former hinds the quaternary
The cholinesterase of our choice is the horse serum hutyryl cholinesterase which is inexpensive and readily available. The method we use is a spectrophotometric one developed by Ellman, Courtney, Andres, and Featherstone (23). This method has been used to study the erythrocyte enzymes (14) and homogenates of rat brain, kidney, lungs, liver, and muscle tissue (24). The principle of the method is the measurement of the rate of production of thiocholine as acetylthiocholine is hydrolyzed. This is accomplished by the continuous reaction of the thiol with 5:5dithiobis-2-nitrohenzoate ion(1) to produce the yellow anion of 5-thio-2-nitrobenzoic acid (11).
60; The reaction with the thiol has been shown to he sufficiently rapid so as not to be rate limiting in the measurement of the enzyme, and in the concentrations used does not inhibit the enzymatic hydrolysis (24). The rate of color production is measured a t 412 nm in a photometer. We have used both a Beckman DB and a Beckman DU converted to a Gilford digital readout spectrophotometer. The results were quite reproducible in each case and suggest that any double beam instrument is satisfactory. It should he pointed out that a difference spectrum is necessary as the disulfide (I) spontaneously forms the anion (11) a t both high and low pH's. A colorimeter may be used providing that separate blank hydrolysis runs are made and the student then manually calculates a difference spectrum. This admittedly increases the error somewhat, Volume 50, Number 9 . September 1973 / 599
0.02
0.02 MNarPzO, (ml)
(3) Each bend in the plot indicates the p K of an ionizing group in one of the reactants, and the straight-line portions intersect as a pH equal to the pK. (4) EachpKcauses a change of one unit in the slope. (5) Each p K of a group situated in the E S compound produces an upward bend with increase of pH; each p K of a group situated in either the free enzyme or the free substrate produces a downward bend. (6) The curvature a t the bends of the plot is such that the graph misses the intersection paint of the neighboring straightline sections by a vertical distance of 0.3 units (log 2); if twwpK's occur together, this distance is0.477 units (log 3). (7) The slope of any straight-line section is numerically equal to the change of charge occurring in that p H range in which the ES compound dissociates to free enzyme and suhstrate. In general, an enzyme-substrate compound with charge n will dissociate to the free enzyme with charge p and the free suhstrate with charge q
M Buffer Solutions at 28'C 0.02 M H ~ P O I (ml)
pH
ES" b u t trial r u n s indicate t h a t such a n approach c a n give meaningful results as well as financial justification. Experimental Procedure The procedure used by our classes involves the following bulk solutions: (a) enzyme solution of 1.0 mg/ml in water which is diluted 20:l with buffer just before use, ( b ) 10 mM suhstrate solution which is varied for Michaelis studies, ( e ) 10 mM thiol reagent in 0.05 M p H 9.0 phosphate buffer to which is added 1.5 mg NaHCO3 per ml of solution. This is good for 4-5 days if stored in the dark a t 4'C. 'The buffer solutions used herein are .02 M phosphate-pyw phosphate. We have determined the pH of a standard scr of a h [ions on a Corning model pH meter using Flsher standard p l l A(,lutions. These data are contained in the table. Four 20 ml beakers are used, and are marked a s sample A and B or reference A and B. Into the beaker marked sample A, the student pipets 5.8 ml of buffer, 1 ml of suhstrate, and 0.2 ml of thiol reagent. Reference A contains 6.8 ml of buffer and 0.2 ml of thiol reagent. Sample B and reference B will each contain 2 ml of buffer and 1 ml of enzyme. Should an inhibitor study be undertaken, the 2 ml of buffer in the sample B beaker e m he replaced by an appropriate amount of an inhibitor such as neostigmine bromide. At time zero, the contents of the A beakers are poured into the B beakers and then these in turn are transferred to cuvettes in the spectrophotometer. The students then take readings every 30 sec for a period of 3 min. Should an inhibition study be attempted, one must allow for a 2 min incubation period in sample heaker B where the enzyme and inhibitor are in immediate contact. This is an important point and should be discussed regardless of whether an inhibition study is undertaken. All the chemicals used in this study were reagent or A grade. These were obtained from either ICN, Nutritional Biochemicals, or the Sigma Biochemicals Corp. ~~~~
~~
~
Z S
EP
+
Sv
The change in slope corresponding to the change in charge will he n-p-q. Yakovlev a n d Agabekyan (29) h a v e studied horse serum cholinesterase using b o t h Dixon's analysis a n d their own, obtaining similar results with each. Using acetylcholine as a substrate, t h e y determined pK1 and pK2 for t h e free enzyme to be 8.85 and 6.1. T h e values for t h e ES complex were determined t o be 9.0 a n d 5.87. K r u p k a a n d Laidler (30, 31) have previously considered t h i s problem i n t h e case of acetylcholinesterase a n d ob-
~
Kinetics Analysis
6
7
8
9
10
pH Figure 1. Plot of pk, (open circles) and log V, pH for butyrylthiocholine iodide at 30%
(dotted circles) versus
The laboratory experiment consists of routine kinetics using Lineweaver-Burke (25) plots as a function of pH. The p H study assumes the Miehaelis (26) relationships for an enzyme with one active form (EH)
Kx = (E-)(H+)/(EH)
(8)
A number of different substrates which give similar results may he used. Dixon (27) has provided a general treatment of pH effects upon K,, the enzyme-substrate constant. These effects are readily extended to K, as fallows (28). (1) The graph of pK, versus p H will consist of straight-line sections if the p K values for the individual reactants are sufficiently separated. (2) These straigbt-line portions will have positive or negative integral slopes of 1.0 or 2.0, zero, or -1.0 or -2.0. 600 /Journal of Chemical Education
6
7
8
9
to
I
pH Figure 2. Plot of pk, (open circles) and log V , (dotted circles) versus pH for acetylthiochoiine iodide at 30°C.
tained p K values of 6.48 and 9.35 for the free enzyme (EH), 6.20 and 10.10 for the ES complex and 6.27 and 10.03 for the acetylated complex. Results
Figures 1 and 2 contain sets of class results for the suhstrates hutyrylthiocholine iodide and acetylthiocholine iodide a t 30°C. By comparing the pKm and log Vm versus p H plots, we are ahle to deduce pK's for the ES complex, the free enzyme and the free suhstrate. In the case of hutyrylthiocholine we determined p K values of 6.4, 6.8, 8.4, and 9.3 from the pKm versus p H plot. The log Vm versus p H plot yields values of 6.4 and 9.3 which are identical with two of the values obtained from the initial pK, plot. The values 6.8 and 8.4 can then he associated with either the free suhstrate, enzyme, or a combination thereof. For reasons which follow, we assign the p K of 6.8 to the hutyrylthiocholine and the value of 8.4 to the free enzyme. The latter may well he associated with a cysteine residue. From Figure 2, we are ahle to determine pK's of 6.1, 6.8, 8.1, and 9.5 from the pK,-pH plot, as compared to 6.3 and 9.4 from the log Vm-pH plot. The values of 6.1 and 9.5 are assigned to the ES complex and are comparable to the values of 6.4 and 9.4 determined with the previous suhstrate. The p K of 6.8 is assigned to acetylcholine and the value of 9.5 is assigned to the free enzyme. The p K of 6.8 for the two thiocholines is a reasonable value as sulfur compounds such as these can he titrated, and ~ e r f o r mas a weak acid. Actual titrations of these compounds yielded p K values of 6.6 for both compounds, makine our choice a reasonable one in view of the criteria outlined in the section on kinetic analysis. The pK's of 6.1, 6.3, and 6.4 can he assigned to the imidazole ring of a histidine residue as was indicated in earlier work (29-31). The oK's of 9.3. 9.4. and 9.5 are intermediate to those reported previously and are difficult to assign. If we are ohserving a single effect, then one is tempted to assign these pK's to a modified tyrosine residue. In any case, this last ~ o i n can t provide considerable speculation on the part of kach laboratory class, and thus becomes a very useful instructional tool. While the above results are encouraging, there are definite limitations in this approach. First of all, the lack of a
unit slope at various pH's suggests that in reality, we are determining an additive pK due to a number oi difierent residues which lie in a complex environment. With this in mind, we suggest that our results are instructive, hut only if the class is informed of their limitations. Finally, we wish to add that the students are recommended t o consult appropriate texts (32) for a more detailed treatment of enzyme kinetics. Acknowledgment
The authors wish to thank the National Science Foundation for the award of grant GY-7748 which provided the necessary equipment funds. Single copies of the actual laboratory experiment are available upon request.
(3~schm'ansohn.D.,'The&ieal and Moleeuhr Beais of Nerve Activity? Academic
,.",",. 191 Milrdi. R.. Malinoff. P.. endPatfer. L. T..Nofure. 229.551 (19711 h~~., [ioi Mefrger.~.P..and W i l s a n . l . B . , ~ i ~ ~3.926(1%4). (11) Prince. A. K.,Amh Biorham. Biophys.. 113.195 11966). (12) Podleaki, T. R..and Nachmansohn. D.,Fmc. Not1 Acod Sci. 55.1034(1966) (13) Auguatinsaon, K. B., Biorhim. Biophya. Aefo. 128,351 (1966). ( 1 4 Reinor. E.. and Aldridee. W. N..Blorhsm. 5 . 105.171 (1967). (15) K7upka.R. M.. Bioch&. 6.1183l19671. (16) Iverson. F.. andMain.A.R..Bioehem., 8.1889l1969). (17) Leuzinger. W . , h &BminRss.. 31.241 (1969). (18) Dauss, D. R., and Green, A. L., in "Advances in Enwmology," Vol. 20, (Editor: Nod. F. F.)lntoncio"ee. 1959. p. 283. (19) Calrmeo,M. H..andEley,D.D.,Biochim. Biophys. Aclo. 58.231 (1962). ( W )O'Bricn,R.D..J. Agr. Food Chem., 11.163l19631. (21) Thomas, J.. and Staniforth, D.,J. Phorm. Phormacal., 16,522 (1964). 122) Hume, A. S., and Holland, W. C., J.Mad. Chem., 7.682 (1964). 1231 Ellman. G. L.. Courtnev. K. D:. Andres. V.. and Featherstone. R. M.. Biackm. P ~ O ~ ~ O C O I 7.68 ~ & 1i9611. 124) Ellman. G.L..Areh. Bioehem. Biophya., 82.70l1959). and Burk, D.,J Amer Chem. Soc.. 58.658 (1934). 115) Lineweaver, H., ," Sprinw-Verlap, 1922. (26) Miehedia, L., "Die W a a r r t o f f m m k ~ n ~ n t t t t i i iBerlin, (n)oixon,~ . , ~ i ~ ~ 55, h ~m11953). ~ . r l . (28) Walter, C.. "Steady State Applications in Enzyme Kinetic.." Ranald Re%% 1965, pp.96-103. (29),Yakwlev. V. A,, and Agabekyan, R. S.,Biochem. (Russian) 32,293 (1961). (3O)'Krupka R.M.. andlaidlor. K. J.. %na. For Soe., 5% 1467 11960). (31) Krupka. R.M.,andLsidler, K.J., %m.For Soe., 56.1477l1960). (32) Mahler. H.R., and Cord-, E. H.. "Wiologieal Cherniaw? 2nd Ed., Harper and Rou,NewYork. 1971.
Volume 50. Number 9, September 1973 / 601
