82
Biochemistry
JOHN P. WOLF, 111, AND CARL NIEMANN
if it competes with the substrate for the acetyl enzyme without completely blocking deacetylation. To overcome the substrate inhibition entirely, as in class A behavior, the inhibitor must have no effect on the deacetylation rate when bound by the acetyl enzyme. CONCLUSION The postulated mechanism for cholinesterase-catalyzed hydrolysis is in agreement with the experimental relationships between the optimum substrate concentration, the optimum velocity, and the inhibitor concentration. The mechanism explains many previously reported experiments (Augustinsson, 1948) in which diverse types of behavior were observed with different inhibitors, different substrates, and different experimental conditions, and with enzymes from different sources (Fig. 5, Table I). It also explains the small noncompetitive component observed with type I1 inhibitors such as tetramethylammonium. It is concluded that when the type I1 inhibitors become bound to the acetyl enzyme they a t most partially block dea-
cetylation. In contrast, deacetylation is prevented when the type I inhibitors, such as cis - 2 - dimethylaminocyclohexanol, become bound to the acetyl enzyme. ACKNOWLEDGMENTS I wish to thank Mr. E. S. Graves for technical assistance and Drs. N. E. Good and D. M. Miller for their criticism of the manuscript. REFERENCES Augustinsson, K.-B. (1948),Acta Physiol. Scand. 15, suppl.
52. Friess, S. L.,and McCarville, W. J. (1954),J. A m . Chem. Soc. 76, 1363. Krupka, R. M.,and Laidler, K. J. (1961),J . A m . Chem. Soc. 83,1445, 1448, 1454, 1458. Main, A. R. (1961),Biochem. J. 79, 246. Wilson, I. B. (1960),in T h e Enzymes, Boyer, P. S., Lardy, H., and Myrback, K., editors, New York, Academic Press, Inc., p. 501. Wilson, I. B., and Alexander, J. (1962),J. Biol. Chem. 237,
1323.
The a-Chymotrypsin Catalyzed Hydrolysis of a Series of Acylated Glycine Methyl Esters* JOHN P. WOLF, 111, AND CARL NIEMANNt From the Gates and Crellin Laboratories of Chemistry, California Institute of Technology, Pasadena, California Received July 26, 1962
The kinetics of the a-chymotropsin catalyzed hydrolysis of twenty-two acylated glycine methyl esters in aqueous solutions a t 25.0", p H 7.90,and 0.50 M with respect to sodium chloride have been investigated. These data and those for the only previously known substrate of this class, methyl hippurate, have been integrated into a theory developed earlier to explain the structural and stereochemical specificity of a-chymotrypsin for trifunctional substrates of this enzyme. In 1952 Huang and Niemann reported that the hydrolysis of methyl hippurate was catalyzed by achymotrypsin and throbgh inhibition studies provided support for their conclusion that this substrate was hydrolyzed a t the same site involved in the hydrolysis of previously recognized substrates of this enzyme. They also proposed that methyl hippurate be regarded as n bifunctional substrate to distinguish it from aqalogous trifunctional substrates which contained a side-chain as an additional structural feature. This distinction soon received experimental support from the more extended inhibition studies of Huang and Niemann (1953) and from those of Applewhite et d. (1958). Applewhite ahd Niemann (1959), and Hein and Niemann (1962). In these investigations it was found that methyl hippurate could be distinguished from representative trifunctional substrates by the character of the inhibition produced, for example, by indole. With trifunctional substrates the inhibition was fully competitive tvherew with the bifunctional substrate it was of the mixed type (Dixon and Webb, 1958). The division of acylated a-amino acid derivatives into the two preceding classes of substrates has recently received further support from the studies of
Wallace, who found that whereas systems containing cu-chymotrypsin and trifunctional substrates are relatively insensitive to the presence of 9-aminoacridinium ion, those containing a bifunctional substrate are substantially activated. It is possible to distinguish substrates of any particular class on the basis of differences in the dependence of reactivity upon the structure of the substrate. Although an impressive amount of information of this kind is available for trifunctional substrates (Hein and Niemann, 1961), this is not the case for bifunctional substrates. To remedy this situation we have studied the a-chymotrypsin catalyzed hydrolysis of twenty-two acylated glycine methyl esters. It is the purpose of this communication to describe these experiments, to present further evidence for the distinction between biand trifunctional substrates, and to interpret the kinetic behavior of these bifunctional substrates in terms of the theory of the stereo- and structural specificity of achymotrypsin developed by Hein and Niemann (1961, 1962) from consideration of the behavior of the analogous trifunctional substrates.
* Supported in part bv a grant from the National Institutes of Health, U. S. Public Health Service. Contribution No. 2865 from the Gates and Crellin Laboratories of Chemistry. t To whom inquiries regarding this article should be sent.
The twenty-two substrates were prepared as described below. All melting points were corrected and all analyses were by Dr. A. Elek.
EXPERIMENTAL
I Wallace, R. A. (1962),unpublished results obtained in these laboratories.
83
Vol. 2, No. 1, dicn.-Feb., 1963
a-CHYMOTRYPSIN AND ACYLATED GLYCINE ESTERS
Glycine Methyl Ester Hydrochloride.-This compound was prepared in 95 % yield from glycine, methanol, and thionyl chloride by the method of Brenner and Huber
t-Butyl&ylglycine Methyl Ester.-t-Butylacetyl chloride was prepared in 74 % yield from 50 g of t-butylacetic acid and 140 g of benzoyl chloride (Brown, 1938). Reaction of 43 g of the acid chloride with 40 g of glycine methyl ester hydrochloride as described for the preparation of propionylglycine methyl ester gave a crude oily product which was distilled a t 107" and 0.5 nun to give a clear viscous liquid which cry&allized on standing overnight a t 4". The final product was a solid, m.p.
(1953).
Acetylglycine Methyl Ester.-Acetylglycine (Herbst and Shemin, 1943) was esterified with methanol and thionyl chloride (Brenner and Huber, 1953). The crude product was distilled at 88" and 0.15 mm Hg and the distillate, which crystallized on standing, was twice recrystallized from diieopropyl ether to give 80% of acetylglycine methyl ester, m.p. 57.0-58.7". Anal. Calcd. for C s H 9 0 a (131): C, 45.8; H, 6.9; N, 10.7. Found: C, 45.7; H, 7.0; N. 10.6. Propionylglycine Methyl Ester.-Glycine methyl ester hydrochloride, 40 g, was dissolved in 75 ml of distilled water saturated with sodium chloride and sodium carbonate. T o this solution was added 150 ml of toluene, and the mixture was homogenized with a Vibro-stirrer and maintained a t 0" in an ice-salt bath. A solution of 18.7 g of propionyl chloride in toluene made up to 100 ml with this solvent was added in 10-ml portions to the preceding homogenized mixture. Solid sodium carbonate was added as needed to keep the reaction mixture alkaline. The mixture was stirred for 15 minutes after the addition of the last portion of acid chloride and transferred to a 500-ml separatory funnel, and the toluene phase was collected and the aqueous phase extracted three times with 100-mlportions of ethyl acetate. The toluene and ethyl acetate extracts were combined, the solvents were removed by vacuum distillation, and the residual oil, 13 g, was distilled a t 110" and 1.5 mm. The distillate crystallized on standing and was recrystallized from diisopropyl ether t o give 55% of propionylglycine methyl ester, long needles m.p. 37.0-38.0". Anal. Calcd. for C6HI1o3N(145): c , 49.6; H, 7.6; N, 9.6. Found: C, 49.4; H, 7.4; N, 9.5. Isobutyrylglycine Methyl Ester.-A solution of 18 g of isobutyric acid and 9 g of thionyl chloride in 60 ml of toluene was refluxed for one hour, and the solution was cooled and made up to 100 ml with toluene. This solution was used to acylate glycine methyl ester hydrochloride by the procedure described immediately above. The crude product, a light oil, was distilled a t 90"and 0.3 mm, and the distillate was allowed to crystallize and the solid recrystallized from diisopropyl ether to give 51% of isobutyrylglycine methyl ester, long needles, m.p. 50.861.6". Anal. Calcd. for C7HI3O3N(159): C, 52.8; H. 8.2; N, 8.8. Found: C, 52.9; H, 8.3; N , 8.7. Piualylglycine Methyl Ester.-Pivalyl chloride was obtained by slowly distilling a solution of 1 mole of pivalic acid dissolved in 3 moles of benzoyl chloride as described by Brown (1938). The acid chloride was used to acetylate glycine methyl ester hydrochloride by the procedure described above for the preparation of propionylglycine methyl ester. The crude pivalylglycine methyl ester was distilled a t 111-112" and 4 mm to give a distillate which crystallized after standing a t 4" for 5 months but which could not be recrystaliized except from the melt. The distilled product, nAD-' = 1.4492, was obtained in 80% yield. z4snd. Caicd. f9r CsHlsOaN(173): C, 55.4; H, 8.7: Y , 8.2.. Found: C, 55.3: H, 8.6; N, 8.2. !::osalt.ryigIycineMethyl %fer.- -This compound was p-epared from isovaieric acid iind glycine methyl ester ilydroriiloride as described "or the anaiogous isohutyrylgiy.JIG 4 methyl ester. Tht! crude product was twice distilled a t 96.5" :iIid cj.3 mm. The final distillate, n2[? = 1.4481 resisted crystallization. Anal. Calcd. for C,HI,O,N (173): C, 55.4; H. 8.7: N 9 I . Found: C. 55.4: H, 8.7; N, 8.0. ~
43.5-44 "
.
A d . Calcd. for CgHI7O3N(187): C, 57.7; H, 9.2; N, 7.5. Found: C, 57.7; H, 9.1; N, 7.5. ChloroaL.etylglycine Methyl Ester.-Chloroacetic acid was converted into the acid chloride by the procedure of Brown (1938). Glycine methyl ester hydrochloride was acylated with chloroacetyl chloride as described for the preparation of propionylglycine methyl ester. The crude product was distilled a t 105" and 0.5 mm to give a distillate which crystallized on standing. Recrystallization of this product from diisopropyl ether gave chloroacetylglycine methyl ester, m.p. 43.6 44.6 ". A d . Calcd. for C5Hs0&C1 (166): C, 36.3; H, 4.8; N, 8.5. Found: C, 36.4; H, 4.9; N, 8.5. Dichlorwcetylglycine Methyl Ester.-Substitution of dichloroacetic acid for chloroacetic acid in the procedure described immediately above gave a crude product in 50% yield. Recrystallization of this material three times from diisopropyl ether gave dichloroacetylglycine methyl ester, m.p. 63.1-64.5'. Anal. Calcd. for C,H,O,NCI? (200): C, 30.0; H, 3.5; N, 7.0. Found: C, 30.2; H, 3.6; N, 6.9. Trichloroacetylglycine Methyl Ester.Substitution of trichloroacetic acid for chloroacetic acid in the procedure described above gave a crude product ih 80% yield. Distillation of this preparation a t 95.6" and 0.2 mm gave trichloroacetylglycine methyl ester, a viscous oil, n:: = 1.4953. Anal. Calcd. for C j H 6 0 a C &(234): C, 25.6; H, 2.6; N, 6.0. Found: C, 25.5; H, 2.5; N, 5.9. Trifluoroacetylglycine Methyl Ester.-Glycine methyl ester hydrochloride (15 g) was acylated with 25 g of trifluoroacetic anhydride in the presence of triethylamine by the procedure described below for the preparation of picolinylglycine methyl ester. The crude product was first distilled a t 49-50" and 0.15 mm and then redistilled a t 68.0-68.5" and 1.1mm to give the desired product as a colorless liquid. Anal. Calcd. for C i H 6 0 8 F 3(185): C, 32.5; H, 3.3; N, 7.6. Found: C, 32.6; H, 3.5; N, 7.4. Ethoxyacetylglycine Methyl Ester.-Ethoxyacetic acid was prepared from chloroacetic acid and sodium ethoxide as described by Rothstein (1932). The acid was converted to the acid chloride by the procedure of Brown (1938) and the latter compound used to acylate glycine methyl ester hydrochloride as described for the preparation of propionylglycine methyl ester. The crude product was distilled a t 97-100" and 0.3 mm to give 80% of product, a viscous oil, n:? = 1.4481. Anal. Calcd. for CjHlaOIN(175): C, 48.0; H, 7.5; N, 8.0. Found: C, 48.2; € I , 7.3; N, 7.9. Ethylmercaptoacetylglycine Methyl Ester.---Ethylmercaptoacetic acid was prepared from chloroacetic acid and sodium ethyl mercaptide by the procedure of Ramberg !1907). The acid chloride was prepared as before I Brown, 1938') and used to acylate glycine methyl ester hydrochloride as in the preceding preparation. The crude product was twice recrystallized from diisopropyl cther to give the ester, m.p. 60.7-62.7", in 80% vield. A - i J m iCalcd. . f
