Aug. 5 , 1957
HYDROLYSIS OF ACETYL-L-TYROSINAMIDE
stituting groups in place of the (CH3)PN- substituent on the azobenzene nucleus of I. A corresponding dye with a para 0-H group, for example, would allow one to study ionization behavior in the region near p H 7-8, an ortho 0-H group, higher CH ranges. One is also not limited to attachments a t mercaptan side chains of proteins. The use of a sulfonyl chloride substituent in place of -HgAc in I, for example, would permit one to make adducts to lysine residues. There is thus a wide range of covalent complexes which can be prepared. These
[CONTRIBUTIOS S O .
2170
FROM THE
GATESAND
4085
should offer suitable examples for establishing how much emphasis should be put on the role of “frozen” water in determining the chemical and physical behavior of dissolved protein molecules. Acknowledgments.-This investigation was assisted by a grant from the National Science Foundation. We also are indebted to Dr. Howard K. Schachman for his suggestions in the use of the synthetic boundary cell. EVANSTON, ILL.
CRELLIN LABORATORIES O F CHEMISTRY, CALIFORNIA INSTITUTE OF TECHNOLOGY]
Further Studies on the Kinetics of the a-Chymotrypsin Catalyzed Hydrolysis of AcetylL-tyrosinamide and of Acetyl-L-tryptophanamide in Aqueous Solutions at 25” and pK 7.9 et 0.11 BY RICHARD A. BERNHARD AND CARLNIEMANN~ RECEIVED JANUARY 25, 1957 I t has been found t h a t the values of Ks, k3 and Kp for the system a-chymotrypsin-acetyl-L-tyrosinamide, in aqueous solutions a t 25’ and PH 7.9 rt 0.1, obtained by observing the rate of formation of either one of the two reaction products, are identical within the limits of experimental error, t h a t t h e values of Kg and Kp are independent of the nature and concentration of the buffer components of the four buffer systems investigated and t h a t the values of k) are dependent upon the concentration of the buffer components and indirectly upon the nature of the buffer species, More limited observations with t h e system a-chymotrypsin-acetyl-L-tryptophanamidesuggest t h a t the behavior of this system is identical with that observed for t h e system or-chymotrypsin-acetyl-L-tyrosinamide. An experimental criterion for the recognition of bi- and trifunctionality in anionic competitive inhibitors of the type RCHzCO2- and R’CONHCHRC02- has been noted.
In a previous study Thomas, Mac1411ister and Niemann3 examined the kinetics of the a-chymotrypsin catalyzed hydrolysis of acetyl-L-tyrosinamide in aqueous solutions a t 25’ and pH 7.9 f 0.1 and 0.02 M i n the THAM4component of a THAMHCl buffer, or 0.02 M in the EDL45component of an EDA-HCl buffer, by determining the rate of formation of acetyl-L-tyrosinate ion. The results obtained by these investigators were re-evaluated by Foster and N i e m a d and by Foster, Shine and Niemann’ and it was concluded that the above reaction systems could be represented throughout their course by equations 1 and 2, where Plf is acetyl-L-tyrosinate ion and P2feither ammonia or amEt
+ SIJkikz_ ES +Er + Pis + Pzr kc Er + Pir I _ EPi ks k3
(1)
(2)
monium ion, and that their rates could be described, within the limits of experimental error, by eq. 3, where Ks = ( k p k 3 ) / k 1 = 32 =t4 X M6,
+
KP = k5/k4 = 110 f 30 X W a n d k3 = 2.4 f 0.3 X M/min./mg. protein nitrogen per ml.6. (1) Supported in p a r t b y a g r a n t from t h e National Institutes of Health, Public H e a l t h Service. (2) To whom inquiries regarding this article should be sent. (3) D. W. T h o m a s , R. V. MacAllister a n d C. Niemann, THIS JOURNAL, 73, 1548 (1951). (4) Tris-(hydroxymethy1)-aminomethane. ( 5 ) Ethylenediamine. (6) R. J. Foster a n d C. h’iemann, THISJOURNAL. ‘77, 1886 (1955). (7) R.J. Foster, H. J. Shine a n d C. Niemann, ibid., 17, 2378 (1955).
When the above values of Ks and ks are compared with those reported earlier,8-11 and which were obtained by determining the rate of formation of ammonia and ammonium ion in aqueous solutions a t 25’ and PH 7.8 and 0.1 M in an unspecified phosphate buffer containing sufficient lithium chloride to bring the initial ionic strength to 0.292, it is seen, cf. Table I, that such a comparison leaves unanTABLE I KINETICCONSTANTS FOR THE a-CHYMOTRYPSIN CATALYZED HYDROLYSIS OF ACETYL-L-TYROSINAMIDE IN AQUEOUS SOLUTIONS AT 25’ AND pH 7.9 et 0.1 Ref. Ks’ krb KP’ 3, 6, 7 32 et 4‘sd 2.44 f 0 . 3 ’ ~ ~ 110 f 30Cmd 8 23”’ 2.76.’ ........ 9 32.6’s.’ 2.7’*’ ........ 10 27”J.’ 3 .o”,f,a ........ 11 29”’ 3.1” ........ a In units of 10-8 M . b In units of 10-8 M/min./mg. protein-nitrogen per ml. =Reaction system 0.02 21.1 in the THAM component of a THAM-HCl buffer or 0.02 M in the EDA component of an EDA-HCl buffer. Based upon the determination of the rate of formation of acetyl-Ltyrosinate ion. Reaction system 0.1 M in an unspecified phosphate buffer containing sufficient lithium chloride to bring t h e initial ionic strength to 0.292. f Based upon the determination of the rate of formation of ammonia and ammonium ion. OSaid t o be preferred to the two sets of values immediately above.
swered a number of questions whose answers are required for a more complete understanding of the reaction under consideration. It is the purpose of (8) S. K a u f m a n a n d H . Neurath, Arch. Biochem., 21, 245 (1949). (9) S. K a u f m a n a n d H. Neurath, J . Biol. Chcm., 180, 181 (1949). (10) G. W.Schwert and S. K a u f m a n , ibid., 180, 517 (1949). (11) S. Kauftqag and R . Neurath, ibid., 181, 623 (1949).
this comniunication to provide answers to several 'r4lnI,K II these questions, i.e., are the same values of I&, ,I CIITXVTKYPSIN CAIALYZEDH Y D R O L YI>11:I ~- ~ C W Y L - I . k3 and Kp obtained when the reaction is examined TTROSISAMIDE I N AQGEOUS S O L U T I O S S A T 25' A X n by observing the rate of formation of both Plf and 7.9=k 0.1 .4xn 0.02JI IN THE T H A M COMPO. P?t, are the values of h-s, k3 and K p derived from TIIAh.T-HC1 BUFFIR studies of the reaction in THAM-HCl and EDL%-[E]: [. I ' ; ii)-driil J HC1 buffers identical with those observed for sys0.222'1 111 1111 73.0 tems containing sodium or potassium phosphate 1'1 I Ill 72.0 buffers and is there any dependence of the values of 2ow 7 122 86.4 K s , k:j and Kp u p ~ the i concentration of the buffer 7 110 71.6 components? 111 110 70.2 A colorimetric procedure for the determination oi 1 ,-I 1.Ti 1 84 2 ammonia, based upon the earlier work of Xoore and L'l I I .i i! 78.6 Stein,12was developed with due consideration being 2 .i 1 .iO 79.7 given to its applicability in systems containing CY:{I 1 165 7 7 ..i chymotrypsin, acetyl-L-tyrosinamide, acetyl-L-tyro:I.; 1I,. I BD.6 sine and THAM hydrochloride as well as ammo4(i 1 su 76.8 nium chloride. This procedure was capable of de1r, I %?(I 65 -4 termining quantities of ammonia, present as ammo.l i V > 110 72.4 nium chloride, of from 0 to 0.G X 21'. 1 1io 67.2 M:ith the above analytical procedure it became l!I 110 66.9 possible to examine the a-chymotrypsin catalyzed 11I 110 62.5 hydrolysis of acetyl-L-tyrosinamide in aqueous solu:j1 I 1 31 ($7.5 tions a t 25" and pH 7.9 + 0.1 and 0.02 Jf in the 41I 1.?I I 65 1 T H A N component of a THAN-HC1 buffer with . 1:1:3e 3 A0 40.0 respect to the rate of formation of ammonia and :i m I 40.i ammonium ion. Thirty-two experiments were con60 :19.7 ducted, c j . Table 11, and it was found that with 5 tj0 :B. 0 I8 the extent of reaction was sufficient to permit the 1 6i I X.(I use of the graphical procedure of Foster and Nie1 60 4 4 .( I niann13--'5for the evaluation of the experimental 10 BO .37 2 data. The above graphical procedure could not be 10 60 38.2 used for the evaluation of the remaining fourteen 1il 61) :%.9 experiments because of a more limited extent of reIO 60 3 7 .6 action. Therefore, in these latter cases the cor10 60 3 0 .0 rected initial velocities were evaluated by the pro.026fje 5 60 8.3 cedure of Jennings and Siemann16-1sand these val10 6iJ 7 2 ues of were then used in an Eadie plot'j to obtain 10 cio 7.6 values of K s and kBbut not of Kp. In units of In units of mg. protein-nitrogen per 1111. Two separate evaluations were performed. I n Maximum time of observation in minutes with the first the experimental data obtained from the a total?if. of seven observations taken within t h e time indicated. ten experiments conducted at [E] = 0.200 mg. d Arrnour preparation no. 00892. e Armour preparation no. protein-nitrogen per ml., cf. Table 11, were pre- 10705. J T h e first 18 experiments were evaluated b y the sented in a ([SI, - [ S ] ! ) / tLIS. (In ([SIo,/[Slt))/ t procedure of Foster and Siemann,13-16 t h e remainder by p l ~ t l ~ -and - ' ~ from this plot the following values t h e procedure of Jennings and nTiemann.16-1R 2 X M, k3 = initial velocities, obtained from either a ( [S]Owere obtained: K s = 34 ?.,? + 0.1 x M'min. 'mg. protein-nitrogen [ S ] LI )t us. (In ([SI" '[SI!))'t plot or b y the procedure per ml. and Kp = 80 =t 20 X M . -4s the of Jennings and Niemann,16-1swere normalized to preceding values were derived from experimental an enzyme concentration of 0.200 mg. proteindata obtained under conditions where [E] = 5.7 X nitrogen per ml. assuming that the initial velocities 10-5 JI.lg E's = [E]' K s = 0.2 X I O p 2 and S'S = were directly proportional to the enzyme concen[SI'Ks= 0.15to 1.28 it follows that all assump- trations. .i line was then fitted t o the 32 values of tions inherent in the evaluation procedure have YO and of V G / [SI0 and from the slope and intercepts of been satisfied.6.' this line and the slopes of the 18 lines of slope In the second evaluation, based upon the results - k - s ( k ' p [ S ] O ) / ( K-P ITS)the following values obtained in all 32 experiments, cf. Table 11, the were obtained; K s = 34 1X -11, k3 = 2..5 f 0.3 X lop3 -U/'inin.~nig. protein-nitrogen per (12) S . Moore a n d \V. H. Stein, J . Bioi. C h e m . , 116, 367 (1948). 126. With [E]beml. and K p = 80 + 20 X (13) R . J . Foster a n d C. Niemann, Proc. Y d . d c a d . Sci., 39, 999 (1953). E's between the limits of 0.76 to 6.3 X (14) T . I]. ( 1 3 ) K. A. Booman and C. Xiemann. i b i d . , 77, 5783 (19551. (14) R.J. Foster and C. Niemann, i b i d . , 77, 1886 (1955). (15) R. J. Tiostpr, H. J. Shine and C. Xiemann, i b i d , 7 7 , 2378 (19~751,
