1012
COMMUNICATIONS TO
bonded was stable to 1 N sulfuric acid and labile to 1 N sodium hydroxide a t room temperature. These features strongly suggested that the unknown acid was a decarboxylation product of pketoadipic acid and that the chlorine was situated on a carbon alpha to the ketone. Assuming a beta decarboxylation, these considerations limited the possibilities to either 8-chlorolevulinic acid or pchlorolevulinic acid. Consequently dimethyl a chloro-@-ketoadipatewas synthesized by the chlorination of dimethyl P-ketoadipate with 1 equivalent of sulfuryl chloride according to the method of Xllihn for the chlorination of ethyl acetoacetate.2 This intermediate was hydrolyzed and decarboxylated in 7 il: sulfuric acid to yield S-chlorolevulink acid (m.p. 73-73.5’). Anal. Calcd. for C5H;Q3Cl: C, 39.85; H, 4.69; C1, 23.55. Found: C, 39.92; H, 4.60; C1, 23.43. The position of the chlorine in the synthetic product was established by displacement of the chlorine with hydroxide ion and then periodate oxidation to yield formaldehyde which was isolated as the dimedone derivative (m.p. 191O). The enzymatically synthesized chlorine containing acid and the synthetic product were identical as shown by constant specific radioactivity on repeated crystallization (synthetic 6-chlorolevulinic acid (24.74 mg.) was added to a purified preparation of the unknown radioactive acid (24,000 f 1,000 c.p.m.) the specific activity (c.p.m./mg.) after successive crystallizations from hexane-ether was 770, 1016, 1081 and 1021). In addition, paper chromatography of the natural and synthetic compounds revealed identical behavior in several solvent systems. Chromatography of the natural and synthetic compounds on silicic acid yielded fractions having a constant ratio of weight to radioactivity. Acknowledgment.-This work was supported by a grant from the Xational Science Foundation.
THE
VOl. 81
EDITOR
conditions where [E] = 4.54 X M” and [SIo, was varied from 5.0 X 10P3 A 1 to 6.0 X lo-‘ dl. When the results of these experiments were pre,~ apparent sented in a D O vs. v O / [ S ]p~ l ~ t ,it~became that the plot consisted of two essentially Iinear segments, of differing slope and intercept, and a nonlinear connecting segment. The slope and intercept of the linear segment associated with values -I1led to of [SI0 = 5.0 X M to 3.0 X values of Ks = 1.0 X M and kI = 1.2 X l o p 4 X/min./rng. protein nitrogen per ml. The slope and intercept of the other linear segment, associated with values of [SIO= 5.0 X l o F 2 JI to 6.0 X lo-’ -If,gave values of ICs = 4.1 X lo-’ M and k I = 1.0 X l o p 3 &‘min./mg. protein nitrogen per ml. For a system represented by equations 1 to 4, inclusive, it can be shown that the rate equation for such a representation is given by equation 5 ki E + S ~ E S
(1)
ko
k, ES+E+P ka
BS + s
(2)
ESr
(3)
kz
ks ES:!+ES f P
2’0 =
((Ks”ks
+ kdSId[El [SIoJ/{Ks”[SIo+ [SI? + Ks’Ks”1
+
(4)
(5)
where Ks’ = (k2 k g ) / k l and Ks” = ( k j -I- k6)/k4. With Ks’ = 1.0 X l o p 2 M , Ks” = 4.1 X lo-’ 111, k3 x 10-4 M/min./mg. protein nitrogen per ml. and k6 = 1.0 x 10-3 kf/min./mg. protein nitrogen per ml. it was found that equation 5 provided a satisfactory quantitative description of the dependence of vO upon [SIoover the 120-fold range of substrate concentration that had been studied. Therefore,. we conclude that the a-chymotrypsin (2) F.B l l i h n , Ber., 11, 587 (1678). catalyzed hydrolysis of methyl aceturate may be CONVERSE MEMORIAL LABORATORIES represented by equations 1 to 4, inclusive, and that HARVARD UNIVERSITY PAULL). SHAW LOWELL P. HACER evidence has been obtained with respect to the forI~EPARTMESTOF CHEXISTRY mation of an ES,complex that is capable of yieldCAMBRIDGE, MASSACHUSETTS ing reaction products a t a greater rate than the I
