1552
ROBERTJ. FOSTER AND CARLNIEMANN
anol and 720 ml. of water was adjusted to pH 8.0 with 1.0 N aqueous sodium hydroxide, 100 mg. of achymotrypsin added and the PH of the reaction mixture maintained at PH 8.0 by the addition of 1.0 N aqueous sodium hydroxide. After 30 minutes a t 25' the reaction mixture was evaporated in vacuo to a volume of 150 ml. whereupon crude I V crystallized from the concentrate. Crude I V was recrystallized twice from aqueous ethanol to give 5.5 g. of IV, m.p. 95-97', [ a ]%D -24.8' (c 7% in ethanol), Anal. Calcd. for C I ~ H ~ I O(251): ~N C, 62.1; H, 6.8; N, 5.6. Found: C,62.2; H, 6.8; N, 5.6. Acetyl-D-tyrosinamide (VI) .-VI, m.p. 225-226', [ a ]%D -49.4' ( G 0.9% in water) was prepared from I V in the same manner and in approximately the same yield as was I1 from 111. Anal. Calcd. for C I I H I ~ O ~(222): N ~ C, 59.5; H, 6.4; N, 12.6. Found: C, 59.7; H, 6.6; N, 12.5. AcetyhAyrosinamide (VII) .-VII, optically inactive, 1n.p. 197-198', was prepared from V in exactly the same way as was I1 from I11 and VI from IV. Anal. Calcd. for C11HlaOsN2 (222): C, 59.5; H, 6.4; N, 12.6. Found: C, 59.4; H,6.4; N, 12.5. Mechanical Separation of Acetyl-D- and L-Tyrosinamides from Acetyl-DL-Tyroshamide.-A preparation of VII, m.p. 197-198', was recrystallized slowly from aqueous ethanol so as to give large well-defined single crystals. The m.p. of any single crystal was found to be 226-228', which is the m.p. of either I1 or IV. The mixed m.p. of a single crystal isolated from the DL-mixture with I1 was found t o be either 197-198' or 226-228', depending upon whether the single crystal selected from the DL-mixture was V I or 11. Enzyme Experiments.-The only departure from the methods described earlier4128was the use of 36-38% Merck and Co., Inc., reagent grade aqueous formaldehyde, adjusted to PH 8.0 with aqueous sodium hydroxide instead of with solid magnesium carbonate.
The precision of the measurements reported in this study was identical with that obtained pre~ i o u s l y . ~Two enzyme preparations, both obtained from Armour and Company, were used in (28) B. M. Iselin and C Kiernann, J Rtol Chcnt , 182, 821 (1950)
[COXTRIBUTION S O . 1468 FROM
THE
VOl. 73
TABLE I11 SUMMARY OF EXPERIMENTS USEDFOR THE EVALUATION OF KINETICCONSTANTS
-
No of ex eriments a t [SP0 1f- 3 molar X SI
I1
[EIQ
5
0 2030
1 1
1 1 3
15
20
30
40
1
1 1 3 1 1 1 1
1
1
y
IC
I1 150d 1 I1 . 13gb 2 2 3 I,$l' I1 :Ig 13gb 1 1 1 I1 :I h . 13gb 1 1 1 1I:IV .13gb 1 '1 I1 :IV'C 13gb 1 1 1 I1 :IT" . 13gb 1" I1 :VI' . 150d 1 1 1 11. VI* . 150d 1 1 In I1 :l . I O .l j O d 1 1 1 11:VI' .150d 1 1 1 Mg. protein-nitrogen per nil. * L o t 110. 90.102 c y = 25. Lot no. 70902. e y = 8. f y = 13.3. [ I l O= [pli0 = 25 x 10-3 M . h [ l i a = ; P ~ I=~40 x 10-3 hi' [ l i O= 5 x 10-3 M . Y = 35. [liO= IO x 10-3 M. [I10 = 15 X M. y = 20.5. y = 45. 0 [ I I 0 = 20 x i o - 8 ~ . p [ l i O= 40 x 10-3 M.
the present study. Of the constants summarized in Table I, those obtained in experiment no. 3 were with lot no. 70902, and all of the others with lot no. 90402 The initial conditions used for the evaluation of the kinetic constants described in this communication are summarized in Table 111. It will be noted that a total of 53 experiments were performed exclusive of those required for the determination of the pH-activity curve and those required for the reactions which were allowed to proceed to near completion. RECEIVED SEPTEMBER 11, 1950
PASADENA 4, CALIF.
GATESA N D CRELLIN LABORATORIES O F CHEMISTRY, CALIFORNIA TECHNOLOGY ]
INSTITUTE O F
The Kinetics of the a-Chymotrypsin-Catalyzed Competitive Hydrolysis of Acetyl-Ltryptophanamide and Acetyl-L-tyrosinamide in Aqueous Solutions at 25" and pH 7 9 BY
ROBERT J. FOSTER AND
CARL
NIEMANN~
An investigation of the kinetics of the a-chymotrypsin-catalyzed competitive hydrolysis of acetyl-L-tryptophanamide and acetyl-L-tyrosinamide in aqueous solutions at 25' and fiH 7.9 has provided independent evidence that these two substrates are hydrolyzed to the corresponding acylated a-amino acids and ammonia via combination a t the same catalytically active site of the enzyme molecule.
The question as to whether two or more substrates do or do not react a t the same catalytically active site of an enzyme molecule can be answered if data are available relative to the reaction kinetics of systems containing the enzyme and substrates, the latter both singly and in competition. In view of the fact that data are now available for the kinetics of the a-chymotrypsin-catalyzed hydrolysis of acetyl-L-tryptophanamide and of acetyl-L-tyrosinamide in aqueous solutions a t 25' and PH 7.W4 the kinetics of the a-chymotrypsin-catalyzed competitive hydrolysis of these two substrates, at 25' and pH 7.9 in aqueous solu(1) Supported in part by a grant from Eli Lilly and Co. ( 2 ) To whom inquiries regarding this article should be sent. J O U R N A L ,73, 1843 (1951). (3) H. T. Huang and C. Niemann, THIS (4) D. W. Thomas. R . V. Machllister and C. Nieinann, i b i d . , 73, 1548 (1951).
tions 0.02 M with respect to a tris-(hydroxymethy1)aminomethane-hydrochloric acid buffer, was investigated in order to determine whether the above substrates were hydrolyzed via combination a t the same reactive site of the enzyme molecule. For reasons given previous1y3s4 the reaction system can be limited to enzyme, substrates and reaction products and the reactions can be formulated in terms of the classical intermediate enzymesubstrate complex theory,6i.e. Er f Sir
ki. 1
kr.1
k2.i ki.2
Er ___----~--
4- Szr I_ ESz k2.:
-
+ I'i.ir + fz'1
(la)
+ Pi.zr -I-Pzi
(Ib)
ESI +J3 k.i.2
Er
(.5) J. H. S.IIaldane, "Enzymes," Lot~gri~aiis--Grcet~, I,oiidon, 1930.
April, 1951
a-CHYMOTRYPSIN-CATALYZED HYDROLYSIS O F ACETYL-L-TYROSINAMIDE
1553
the apparent Michaelis constant
where [E] = molar concentration of total enzyme [E!] = molar concentration of free enzyme [SI]; [&I = molar concentration of total respective substrates [Sir]; [&r] = molar concentration of free respective substrates [ESJ; [E&] = molar concentration of enzyme-substrate complexes [Pl.lr]; [PI.zI] ; [Pzf] = molar concentration of free hydrolysis products
Since in all of the experiments reported in this communication the extent of hydro1 sis of either of the two substrates was less than Z O k , the known inhibition of the hydrolytic reactions by the hydrolysis products3t4was justifiably ignored in the development of the rate expressions for the competitive hydrolysis of the two substrates. For the case where all of the reactants possess unit activity it follow^^-^ that [Ell [Sitlk~i= [ESil(kz.i
+
ka.1)
(24
[Et] [S2flki.z = [ES2l(Kz.z
+
k3.z)
(2b)
and
With experimental conditions selected so that [Ef] A ([E] - [ESII - [ES?]),[SI] 6 SI^] and [Sz]* [ S P f ]it can be shown that when
and the apparent rate constant
From equations 8 and 9 i t is seen that the reciprocal of the apparent Michaelis constant KST is the weighted average of the reciprocals of the individual values of Ks1 and Ks, and that the apparent rate constant kaT is dependent not only upon the values of and but also upon the values of [SIo/ Ks for the respective substrates. The apparent KST and k3-r values for the a-chymotrypsin-catalyzed competitive hydrolysis of acetyl-L-tryptophanamide and acetyl-L-tyrosinarnide a t 25' and pH 7.9 were determined experimentally for two different ratios of the competing substrates and these data are given in Table I along with the previously determined kinetic constants of each of the above substrates and those calculated for the competitive case from equations S and 9. Because of uncertainties in respect to the equivalent weight of a-chymotrypsin the concentration of the enzyme is given in mg. of protein-nitrogen per ml. TABLE I WCHYMOTRYPSIN-CATALYZED COMPETITIVE HYDROLYSIS OF ACETYL-L-TRYPTOPHANAMIDE AND ACETYL-L-TYROS~NAMIDE~ Mole Mole . ~ o/, . ,-~ . ~- o/, acetyl+ acetyl-Ltryptophan- tyrosinamide amide ~~
Exptl.
Calcd.
Exptl.
kic
Calcd.
100 0 5.3d O.& 9.0 9.1 0.86 0.78 50 50 25 75 13.8 13.9 1.3 1.2 0 100 30.5' 2.4' " I n aqueous solutions a t 25" and PH 7.9. * I n units of 10-8 M. In units of 10-8 M/min./mg. protein-nitrogen/mI. Cf. ref. 3. ' Cj. ref. 4.
It will be noted that the experimental values for kaT are slightly greater than the calculated values. This discrepancy is not unexpected and Equation 4 is similar in form t o the classical equa- arises from the difficulty of estimating the initial tion ordinarily used fur the evaluation of Ksl8 velocities from either zero or first order plots of the total substrate concentration versus time when the a.e. ratio of the two substrates is changing with time. 1 1 1Ks From equations 3a and 3b it follows that (5) vo [SI0 v
-=v+--
Therefore for a plot of ~ / V T O versus 1/[ST]O !.e., for initial velocities and substrate concentrations it follows that as [sT]O+00 the intercept
the slope
(6) H Lineweaver and D. Burk THIS JOURNAL, 66, 658 (1934).
and for the system under consideration that the ratio of the rate of hydrolysis of acetyl-L-tryptophanamide to that of acetyl-L-tyrosinamide is 1.2 times the ratio of their concentrations. However, for all values of [ST] encountered in the present investigation the total rate of hydrolysis increased as the ratio of acetyl-L-tryptophanamide to acetylL-tyrosinamide decreased as will be seen from the data given in Table 11. Therefore, because of the contributions arising from a change in the ratio of the substrate concentrations, the experimental value of k l will ~ be subject to a positive error and
ROBERTJ, FOSTER AND CARLNIEMANN
I554
VOl. 73
TABLE I1 experimental methods do not permit the use of the THERELATIVEVELOCITIES OF HYDROLYSIS OF ACETYL-L ideal procedure wherein kaT is determined under 1 KTPTOPHANAXIDB ANI, ACE, 1 I'I,-L-TYROSINAMIDEn
ST]^^ 20
Acetyl I trsptophanamirle r r rbf [ S T ] ~e> of t ro
.4cetyl-r
-
tyrocinamide 0;
of [STio
7 of
tilo
'rota1 r elnti \.e velocity
V? 2;, 29 I r) Til 1 26 60 23 "Y 73 i1 1 Til 20 50 55 50 45 1 00 60 50 55 50 45 1 2s a Calculated from equation 16 for the extreme limits of concentration encountered in the competitive case. IJ I n units of 10-3 M .
will be found to be greater than the true value. I t is unfortunate that the limitations of present I
I
I
1
I
-
9 0
8 -
-
conditions where there is no change in the initial ratio of the concentration of the competing substrates. From the data presented in this communication there can be little doubt that acetyl-L-tryptophanamide and acetyl-L-tyrosinamide are hydrolyzed v i a combination a t the same catalytically active site and i t appears reasonable to assume, in the absence of definite proof, that other substrates of the general formula RCONHCHR1CONH2, where R1 = a P-indolylmethyl group or a p-hydroxybenzyl group, will behave similarly. It will be noted that the above conclusions derived from kinetic studies are in accord with the independent observation that a-chymotrypsin probably possesses but one catalytically active site per mole~ u l e . ~ ~ ~ The authors wish to express their appreciation of the assistance rendered by Dr. H. T. Huang during the course of this investigation.
Experimentalg
0
-
-
3 I IO
I e0
1
I
I
30
40
50
Acetyl-L-tryptophanamide (I).3-Ammonolysis of 10 g. of acetyl-L-tryptophan methyl ester gave 8.0 g. of I, m.p. 190-191", after recrystallization from water and drying in vucuo over phosphorus pentoxide. A mixed m.p. of I with an authentic specimen of I3 showed no depression; [CY]% $20.5 * 0.5" ( 6 , 2% in methanol). Acetyl-L-tyrosinamide (11) .LAmmonolysis of 10 g. of acetyl-L-tyrosine ethyl ester gave 4.1 g. of 11, m.p. 225.5226" after recrystallization from methanol and drying in vacuo over phosphorus pentoxide. The m.p. of I1 was not depressed when mixed with an authentic specimen of I14; [a] 2 6 50.7 ~ * 0.5" (c, 0.8% in water). Procedure.-The methods used have been described previously.3~lO The a-chymotrypsin was an Armour preparation (lot no. 90402) and in all experiments the enzyme concentration was maintained at 0.208 mg. of protein nitrogen per ml., a concentration leading to a maximum value of 23'8 of 0.91 X IO-* thus placing all systems in zone A of Straus and Goldstein.11,12 All experiments were conducted a t 25' and pH 7.9 in aqueous solutions 0.02 M in respect to a tris- (hydroxymethyl) -aminomethane-hydrochloric acid buffer. The reduced primary data are summarized in Fig. 1. PASADENA 4, CALIF. RECEIVED SEPTEMBER 11, 1950 (7) E. Jansen, M. D. Fellows-Nutting, P . Jsng and A . K. Balls,
J . Bid. Chcm., 179, 189 (1949). (8) E. Jansen, M. D. Fellows-Nutting and A . K. Balls, ibid., 179, 201 (1949). (9) All melting points are corrected. (IO) B. M. Iselin and C.Niemann, J . Bid. Chcm., 182, 821 (1950). (11) 0.H.Straus and A. Goldstein. J . Gcn.Physiol., 26, 559 (1943). (12) A. Goldstein, ibid.. 27, 529 (1944).
