K. LIJTWACK, H. F. M O W E RAND C. NIEMANN
5690 [CONTRIBUTION
NO. 2171
T‘ol. 70
PROM THE GATES AND CRELLIN LABORATORIES O F CHEMISlRY, CALIFORNIA INSTITUTE O F TECHNOLOGY]
The a-Chymotrypsin-catalyzed Hydrolysis of a Series of Hydrazides. 11. Evaluation of the Kinetic Constants for Aqueous Systems at 25’ and at the Optimum pH for Each Specific Substrate‘ B Y RALPHTdUTUrACK,2 H O W A R D F.M O W E R 3 AND CARL N I E M A N N 4 RECEIVED J A N U A R Y 26, 1957 The dependence of the a-chymotrypsin-catalyzed hydrolysis of L-tyrosiiihydrazide and of six ol-N-acylated-r~-tyrosinhydrazides, in aqueous solutions a t 25‘ and 0.02 ill in the THAM component of a THAM-HC1 buffer and at the optimurn pH for each specific substrate, upon the initial specific substrate concentration has been detertnined. A comparison of t h e values of Ks and ks for a-N-formyl-, a-N-acetyl- and u-I\’-nicotinyl-~-tyrosi~~llydrazide with those for the corresponding acylated-L-tyrosinamides has shown that while the values of KSfor the members of any given amidehydrazide pair may bc approximately equal and the value of ka for the amide member may be greater than t h a t for the hydrazide member of a particular pair, the ratio of the values of ks for each amidehydrazide pair varies with t h e nature of the acyl moiety presetit in the three pairs.
three plots based upon equation 2,9,10 each set of values of vo and [SI0 were fitted by the method of least squares to the line l/ao = b / [ S ] o a , where b = Ks/ka [E],a = l/k3 [E] and b/a = K s . ~The values of a, b, k3/Ks, Ks and ka so obtained are given in Table I along with the experimental pnrameters characteristic of each set of experiments, e.g., pH, [E] and [SIO. Since values of K s and k3 acquire significance only when the ratios E’s = [ E ] / K s and S’s = [S]o/Ks lie within certain limitsI1l values for these latter constants also are given in Table I. If it is assumed as before” that the molecular weight of monomeric a-chymotrypsin is 22,000, its nitrogen content is 16.0%, the monomer possesses but one catalytically active site per molecule and that all of the enzyme is present in the reaction systern as the monomer, it is clear that all values of Es’ given in Table I are well below the value of 0.6 associated with an experimental error of f5yo.” Therefore, insofar as values of Es’ are concerned, all values of Ks and ka were obtained under the conditions required if equation 2 is to have validity. l1 ki k3 If equation 2 is to be used for the evaluation of Ef Sf ES ---f Ef Plf 1’2f (1) K s and k3 and the experimental error is *t5%, it is kz rates, during the initial stages of reaction, may be required that values of S’S be between the limits described, within the limits of experimental error, of 0.05 and 20.” I t will be seen from Table I that by equation 2, where K s = ( K p k 3 ) / k l . There- the values of S’s for a-N-formyl- and a-N-dichloroacetyl-1,-tyrosinhydrazide are well within the above. 210 = -d[Sl/dt = ki[EI[SI/(K. [SI) (?) limits. However, in all other cases the lower values fore, in the evaluation of the constants ICs and k3 of 3’s are either less than or close to the lower h i i t the primary data, ;.e., extent of reaction vs. time, of 0.05 noted above. were first submitted to a first-order c o r r e ~ t i o n ~ ~With ~ L-tyrosinhydrazide the limited solubility, in order to obtain linear rela tionships between ex- i.e., cn. 5 X 10-3M, coupled with a value of K S = tent of reaction and corrected time. From these cn. G X 10-3M makes it unlikely that K s and k:{ linear relationships a least squares fit was employed can be evaluated with greater precision than tliat to obtain values of VO. Since it was observed that attained in the present study unless the experithe values of 2’0 and [SI0 for each specific substrate mental error can be reduced to below f lY0. With the values of Ks gave apparent linear relationships in any one of the a-N-benzoyl-L-tyrosinhydrazide and k 3 given in Table I are suspect for two reasonr. (1) SupportPd i n p a r t by a g r a n t from t h e Natinual Institutes ol H e a l t h , Public Health Service. First, they were obtained under conditions where (2) Dow Chemical Fellow 1952--1953, General Electric Fellow the estimated values of S’S were so low that the 1953-1954. only constant of possible significance is the ratio (3) Shell Fellow 1954-1965. k3/Ks.I* i\rhile it may appear that the situation (4) To whom inquiries regardinq this article should be Sent ( 5 ) Tris-(hydroxymethy1)-aminnmethane. could be improved to a modest degree by eliniinat-
It is known that L-tyrosinhydrazide, a-Nformyl-, a-N-acetyl-, a-N-trimethylacetyl-, a-Ndichloroacetyl-, a-N-benzoyl- and a-N-nicotinylL-tyrosinhydrazide are hydrolyzed in the presence of a-chymotrypsin and the optimum pH for the hydrolysis of each of the above specific substrates, in aqueous solutions at 25” and 0.02 M in the THAM5 component of a THXM-HCI buffer, has been determined.6 In this communication we shall be concerned with the determination of the dependence of the rate of the a-chymotrypsin-catalyzed hydrolysis of the above specific substrates upon the initial specific substrate concentration when such systems are examined in aqueous solutions at 25” and 0.02 M in the THAM component of a THXM-HC1 buffer and in the immediate region of the pH optimum of each specific substrate. It will become evident from the results of this study that insofar as dependency upon the initial specific substrate concentration is concerned, the initial stages of the a-chymotrypsin-catalyzed liytlrolysis of the above specific substrates may be represented by equation 1 and that their respective
+
*
+
+
+
+
+
( t i ) R . Lutwack, H. k’ hfower and C Niemann, TEISJ U U K N ~ I .79, , 2179 (1957) (7) R. R Jennings a n d C Kiemann, ibid.,75, 46x7 (1963:. (8) W. E.M . Lands and C. Niemnnn, ibid., 77. 0.508 (19K5).
(9) H. Lineweaver a n d D. Burk, ibid., 56, 658 (1934). (10) G. S. Eadie, J Bioi Chsm., 146, 85 (1942). (11) R J. Foster and C. Niemann, THISJ O U R N A L , 77, 188G (1955).
CY-CIIYMOTRYPSIN-CATALYZED HYDROLYSIS OF HYDRAZIDES
Nov. 5, 1057
%
wrbmi300t-t0"'" Rid
C43bdb
88888 bSo c c.0 c
.
. .
0. 0 0. 0
0
.
560 1
ing the data obtained with values of [SI0 from 0.06 to 0.12 X M and basing an evaluation upon the data obtained with values of [SI0 from 0.16 to 0.40 X M,12 i t should be noted that in all cases the extent of reaction was between 53 and 83%. This latter situation coupled with the probability that the value of K,,i.e., the enzyme-product dissociation constant, may be of the order of M creates the second uncertainty in that the use of equation 2 under these circumstances could lead to erroneous values of K s and k3. Since the need for observing the reaction under consideration for a longer than optimal period was a consequence of the very low solubility of the specific substrate, which could not be improved except by the undesirable practice of changing the solvent system, it was decided to make the best of a dificult situation by studying the hydrolysis of a-N-benzoyl-Ltyrosinhydrazide in competition with that of a - X acety1-L-tyrosinhydrazide.l3 The experimental data so obtained were subjected to a graphical analysis based upon consideration of all three plots derived frofn equation 2, and the following values were obtained : ks,/Ks, = 0.10 min./mg. proteinnitrogen per ml.; k3, = 0.15 X M/min./mg. protein-nitrogen per ml. and Ksr = 1.5 X M. From these values and the values of Ks, = 2 2 X l o d 3 M , k3,/Ks, and ks,/Ks, = 0.029 and 0.244 min./mg. protein-nitrogen per ml., respectively, it follows that Ks, = 0.5 X M and k3, = 0.12 X M/min./mg. protein-nitrogen per ml. While no great reliability can be placed upon these latter values, it can be concluded that the value of K s is of the order of M and ks of the order of M/min./mg. protein-nitrogen per i d . It is clear that the relationship between the probable values of K s and k3 and the solubilities of L-tyrosinhydrazide and a-N-benzoyl-L-tyrosinhydrazide are such as to preclude a precise evaluation of K s and k3 for these specific substrates. The relatively low lower limits of S'S associated with the evaluation of K s and k3 for a-N-acetyland a-N-triniethylacetyl-L-tyrosinhydrazide suggests the desirability of a re-evaluation of the kinetic constants of these specific substrates a t higher values of [SI, if constants of greater reliability are to be obtained. However, it is known that the values of K s and k3 given in Table I for a-N-acetyl-L-tyrosinhydrazide are in substantial agreement with values so determined. l 4 Since it is possible to examine a-N-trimethylacety1-Ltyrosinhydrazide under conditions where S'S can be varied between the limits of 0.05 and cu. G.0,'6 in spite of the relatively high value of K s , this spccific substrate has been selected for an investigation (12) Such an evaluation was found to give a value of k s / K s = 0.230 =t0.006 min./mg protein-nitrogen per ml. (13) R . J . Foster and C . Niemann, THISJ O U R N A L '73, 15.52 (1831). (14) R . J. Kerr, P h . D . Thesis, California Institute of Technology,
Pasadena, California, 1957. (15) The association of the relatively high melting point, i . e . , 227-22S0, with the limited solubility, ;.e., ca. 0.015 A4 in aqueous solutions at 2 5 ' , of a-N-acetyl-L-tyrosinhydrazide suggested that a more soluble neutral specific substrate derived from L-tyrosinhydrazide could he obtained if the lattice energy of the crystal could be decreased by the introduction of a bulky acyl moiety. a-N-Trimethylacetyl-Ltyrosinhydrazide was prepared and not only was its melting point, i e . , 179-180°, approximately 50° lower than that of a-N-acetyl-& tyrosinhydrazidea but !ts solubility in water at 25' wa- ca. 0 . 2 4 M .
R. LUTWACK, H . F. MOWERAND C. NIEMANN
5692
of the nature of the rate equation for both the initial and extended reaction. These studies, which also should lead to more precise values of K s and k3, are now in progress. When the experimental data for a-N-nicotinylL-tyrosinhydrazide were evaluated, i t was found that only the constants b and k3/Ks could be obtained. The least squares treatment led to a negative value for a. However, a subjective graphical estimate, based upon uncorrected initial velocities, gave the values of Ks and k3 presented in Table I. Although the k3/Ks ratio, ;.e., 0.11 rnin./mg. protein-nitrogen per ml., derived from these values is in agreement with the value of k3/Ks obtained by the least squares treatment, i t is impossible to assign limits of variability to the values of K s and k3 given in Table I. However, they may be accepted as being substantially correct. l 4 Of the seven compounds considered in this study, it is clear that five, ;.e., a-N-formyl-, a-K-acetyl-, a-N-trimethylacetyl-, a-X-dichloroacetyl- and a-N-nicotinyl-L-tyrosinhydrazide are useful specific substrates of a-chymotrypsin. The ratio k 3 / K ~ is an index of over-all reactivity when [SI0 loses significance with respect to Ks. The fact that values as low as 0.0032 and as high as 0.244 could be determined illustrates the exceptional sensitivity and flexibility of the analytical method that may be used for following the hydrolysis of the above specific substrates. .i\L comparison of the values of K s and k3 for a-X-formyl-, a-N-acetyl- and cr-N-nicotinyl-Ltyrosinhydrazide with those for the corresponding amides which were determined under comparable conditions,ll reveals that the values of K s for a given amide-hydrazide pair are approximately equal for all three pairs and that the value of k3 for the amide member of a given pair is greater than that for the hydrazide member of the pair, cf., Table 11. However, when the ratios of k3 for a TABLE I1
Ks AND k3 VALUESOF
AMIDE-HYDRAZIDE PAIRS'
Ksb l c y l moiety
a-X-Formyl a-N-Acetyl a-N-h-icotinyl
Amided
Hydrazide
Amided
knC Hydrazide
9 8 i 0 5 0 45 =k 0 05 0 058 f 0 002 i:O 2 i: 8 2 4 i0 3 0 7 32 i 4 28 12 f 3 9 5 0 + l O 1 12
3
a For L-tyrosine derivatives in aqueous solutions at 25" and a t the optitnuin pH. In units of JI. In units of 10-3 l i / m i n . / m g . protein-nitrogen per ml. Cj., ref. 11.
given pair are compared, it is seen that this ratio is ca. 8 for the a-N-formyl derivatives, ca. 3 for the a-N-acetyl derivatives and ca. 5 for the a-Knicotinyl derivatives. Thus, even when the values of Ks are approximately the same for an ainidehydrazide pair, which differ only in one being an amide and the other a hydrazide, the differences in the values of k3 for such pairs are still dependent upon the nature of the acyl moieties present in the different pairs. IVhile a rigorous and quantitative explanation of this phenomenon cannot be given a t this time, we believe that it may be a consequence of a situation where Ks is an index of the extent of combination of the enzyme and specific substrate in a variety of inodes only some of which lead to orientations in the ES complex which permit the
Vol. 79
formation of a transition state and where k3 is an index not only of the intrinsic energetics of the reaction involving the potentially hydrolyzable bond and its adjacent atoms but also of the probability of combination in a mode which can lead to a transition state.16-18 The values of K s and k3 obtained for a-N-dichloroacetyl-L-tyrosinhydrazide were unexpected since earlier studies with acetyl- and chloroacetylL-tyrosinamide, l1 conducted under comparable conditions, indicated that replacement of an acetyl group by a chloroacetyl group had little or no effect upon the values of Ks, i.e., 32 4 and 27 f 2 X 21; but caused a marked increase in the value of k 3 , ;.e., 2.4 + 0.3 and 4.0 5 0.2 X M/inin./mg. protein-nitrogen per nil. IVith the observation made in this study that replacement of an acetyl group by a dichloroacetyl group in the case of a-N-acylated-L-tyrosinhydrazides has the effect of causing no substantial change in the value of k3, ;.e., 0.7 i. 0.2 and 0.70 + 0.07 X M/ min./mg. protein-nitrogen per nil., but instead of causing a marked decrease in the value of K s , ;.e., 22 =t8 and 5.2 i. 0.7 X l o p 3111,it is clear that until the constants for the systems a-chyinotrypsindichloroacetyl-L-tyrosinamide and a-chymotrypsin-a-K-chloroacetyl-L-tyrosinhydrazide are cletcrmined, it will remain unknown as whether the results noted in this study are a consequence of a unique behavior associated with the presence of the dichloroacetyl group or of the carbohydrazide group or of a combination of both. The authors wish to express their indebtedness to Drs. R. 31. Bock, J. T. Braunholtz, R. Kerr and W. E. 11.Lands and to Mr. Charles Goebel for their assistance during the course of this investigation. Experimental
*
Specific Substrate Stock Solutions .-The specific substrates and their stock solutions were prepared as described pre~iously.~ Buffer and Enzyme Stock Solutions.-The solutions were prepared with the same precautions as before6 from T H X M and crystalline bovine a-chymotrypsin, Xrmour preparations nos. 1OiO5 and 00592. Analysis of Reaction Systems.-The procedure described by Lutwack, Mower and Niemannfiwas used without rnodification. Reaction Systems .-The general procedure has been tlescribed previously .6 All experiments were conducted in aqueous solutions a t 25" and 0.02 AI in the TH.1R.I component of a TH;\M-HCl buffer. The reaction conditions employed with each specific substrate are summarized below. L-Tyr0sinhydrazide.---Ele\-en experiments with values of [SIofrom 0.72 t o 4.12 X If, [ E ] = 0.208 nig. proteinnitrogen per ml. of .Irmour preparation no. 10705, total reaction time of 4*5t o 195 niin., total extent of reaction of 3.0 t o 12.7% gave 11 corrected values of uo of 0.441 t o 1.904 X X,"iii. with each value being determined by a least squarrs fit of A t o 9 points corresponding t o the same number of observations of the currected extent of reaction7~* us. time within the above total reaction times. a-N-Formyl-L-tyrosinhydrazide.-Xiti c experiments rvitli M, [ E ] = 0.1414 values of [SIafrom 3.71 t o 13.17 X tng. protein-nitrogen prr nil. of Xr:nour preparation no. 00592, tot:il reaction tiine of 32 t o 38 rnin., total extent of rraction of 1.2 to 2.iyo gave 9 corrected values of 1'0 of
Nov. 5, 1957
DL-4-OXALYSINE, A N INHIBITORY
2.330 t o 4.940 X M l m i n . with each value being determined by a least squares fit of 8 t o 9 points. a-N-Acetyl-L-tyr0sinhydrazide.-Fifteen experiments with values of [SI0 from 1.27 t o 6.67 X M, [E] = 0.208 mg. protein-nitrogen per ml. of Armour preparation no. 10705, total reaction time of 13 to 55 min., total extent of reaction of 5.8 t o 28.5% gave 15 corrected values of TJO of M l m i n . with each value being deter0.735 t o 3.117 X mined by a least squares fit of 7 t o 8 points. a-N-Trimethylacetyl-L-tyr0sinhydrazide.-Two sets of six experiments with values of [SI0 from 0.494 t o 1.08 X iZI a n d 2.99 t o 10.48 X M , [EJ = 0.1444 mg. protein-nitrogen per ml. of Armour preparation no. 00592, total reaction time of 32 t o 60 min. a n d 16 t o 36 min., total extent of reaction of 6.2 t o 8.670 and 1.9 t o 4.47, gave 12 corrected values of erg of 0.071 t o 1.380 X M l m i n . with each value being determined by a least squares fit of 8 to 9 points. a-N-Dichloroacetyl-L-tyrosinhydrazide.-Ten experiments with values of [SI0 from 0.909 to 3.82 X l o w 3AI, [ E ] = 0.1444 mg. protein-nitrogen per ml. of Armour preparation no. 00592, total reaction time of 9 to 10 min., total extent of reaction of 10.0 to 15 0 % gave 10 corrected Mlmin. with each values of 00 of 0.145 to 0.420 X value being determined by a least squares fit of 7 t o 9 points. a-N-Benzoyl-L-tyr0sinhydrazide.-Two sets of cxperiJI ments, one with 8 values of [SI"froin 0.06 to 0.12 X and the other with 10 values of [SI0 from 0.16 t u 0.402 X l0-3il.i, [ E ] = 0.208 mg. protwi-nitrogen per i d . of -\miour preparation 110. 1070.5, total rv i-ti,iri tiinc of 15 to 27 min.
ANALOGOF LYSINE
5693
a n d 15 t o 39 min., total extent of reaction of 57.1 t o 80.1% and 53.4 t o 82.7% gave 18 corrected values of TJO of 0.290 t o 1.862 X Mlmin. with each value being determined by a least squares fit of 8 t o 9 points. T h e conditions used in the competitive hydrolysis of a-iY-acetyl- and a-N-benzoyl-L-tyrosinhydrazide are summarized in Table 111.
TABLE I11 a-CHYMOTRYPSIN-CATALYZED COMPETITIVE HYDROLYSIS OF a-N-ACETYL-A N D a-N-BENZOYL-L-TYROSINHYDRAZIDE~ [silo [SZlQ [ST10 lmax vo b X 103 M
X 105 M
X 108 M
(min.)
X 106M/min.
0.140 0.268 0.408 14 0.696 .140 .498 14 .762 .358 .140 .447 .487 11 .918' a In aqueous solutions a t 25" and pH 7.9 and 0.02 M in the T H A M component of a THBM-HC1 buffer with [ E ] = 0.208 mg. protein-nitrogeii per ml. of Arnmour preparation no. 10705. From a least squares fit of 7 points unless 8 points. otherwise noted.
a-N-Nicotinyl-L-tyrosinhydrazide .-Thirteen experiments with values of [S]Ofrom 0.50 to 4.56 X M , [E] = 0.208 mg. protein-nitrogen per ml. of Armour preparation no. 10TO5, total reaction time of 7 to 30 min., total extent of reaction of 13.1 t o 47.8Yc gave 13 corrected values of o~ of 0.102 to 0.808 X M/min. with each value being determined by a least squares fit of 7 t o 9 points. PASADENA, CALIFORXIA
FROM THE CLAYTOY ~ ' O U N U A T I O N FOR RESEARCH, THEBIOCHEMICAL INSTITUTE AYD THE DEPARTMEYT O F CHEMISTRY, THEUNIVERSITY O F TEXAS]
[CONTRIBCIIO\
DL-4-Oxalysine, an Inhibitory Analog of Lysine BY TOMMY J. NCCORD, JOANNE M. RAVEL, CHARLES G. SKINNER AND WILLIAM SHIVE RECEIVED JUNE 14, 1957 2-Xmino-3-(P-aminoethoxy)-propionic acid (4-oxalysine) has been prepared through a n acetamidomalonic ester condensation using 2-chloroethyl chloromethyl ether, followed by ammonolysis and hydrolysis of the intermediate, to give a mixture of products. T h e reaction mixture was separated into several pure components by chromatography on an alumina column. 4-Oxalysine inhibits the growth of a number of microorganisms. T h e reversal of this toxicity by lysine has been most extensively studied with Leuconostoc dextranicum 8086 a n d was found to be competitively reversed over a 300-fold range of concentrations with a n inhibition index from 1 t o 3.
The introduction of an oxygen atom in place of a methylene group in the carbon skeleton of a naturally occurring amino acid has resulted in the formation of an inhibitory In the present study, it was desired to prepare an analog of lysine by the introduction of an oxygen atom in place of the methylene group a t the 4-position. Such an analog has the structural similarity and similar distance between the amino groups which would be necessary for antagonism of lysine. For example, 2,6-diaminoheptanoic acid (e-C-methyllysine) has been reported to be an effective lysine inhibitor, but other compounds of similar structure but having amino groups different in distance apart from that of lysine are ineffective as inhibitor^.^ Accordingly, 2-amino-3-(B-aminoethoxy)-propionic acid (4oxalysine) was prepared and found to be an effective inhibitory analog of lysine for several microorganisms. (1) N. H . Horow-itz and A . A I . Srb. J . Bioi. Cheni., 174, 371 (19.18). (2) M . Rabinovitz, X. E. Olson and D . &I. Greenberg, THIS J O U R N A L , 7 7 , 3109 (1955). (3) C . G . Skinner. T. J McCord, J . R I Ravel and \V. S h i r e , i b i d . , 7 8 , 2412 (19.56). (4) A . D. McLaren and C. A . K n i g h t , 5 . Bioi. C h e v . , 204, 417 (1953).
In all of the synthetic approaches attempted, which resulted in the formation of 4-oxalysine, a mixture of products was obtained which proved difficult to separate. Further, the yield of byproducts in every case exceeded that of the desired compound. The most satisfactory technique found was through an acetamidomalonic ester condensation with 2-chloroethyl chloromethyl ether. The desired product of this condensation, 2 - acetamido - 2 - (p - chloroethoxymethyl) - malonate, was always contaminated with unreacted starting material and only a 38% conversion5 was obtained. Several attempts to improve this yield by using inverse addition, altering the temperature and/or time of addition, using other condensing agents6 and by direct fusion of the reactants a t elevated temperatures' failed. The substituted malonic ester derivative was treated with concentrated ammonium hydroxide a t room temperature for several days to replace the terminal chlorine by an amino- group and then acid hydrolyzed; or ( 5 ) T h i s factor takes into account t h e recovered starting material. (6) J. Shapira, R. Shapira a n d K. Dittmer, THIS JOURNAL, 768 3 l i X (1953). ( 7 ) S. SIaeda, SI. Terumi and 7 . Suzuki, Bzrli. l i z s f . P h y s . Ciiem. Reseoi,cii ( T o k y o ) 19, 267 (1938); through C. A , , 34, 6931 (1940).
