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Vol. 76
of the four separate isomers of a p-hydroxy-a-amino acid by this enzyme has not been reported. This is herein accomplished for P-phenyl~erine.~The cleavage of p-phenylserine is of additional interest due to the structural and possible metabolic relationships of the compound with epinephrine and chloroamphenicol. When a purified rat liver preparation was employed as enzyme source, i t was found (Table I) that erythro-p-phenyl-~-serinewas rapidly cleaved a t nine times the rate of threo-p-phenyl-L-serine. The respective D isomers were not measurably attacked under the conditions employed. This result regarding the threo isomers of P-phenylserine is analogous to the data obtained using racemic and Lthreonine which indicated that L-threonine was selectively a t t a ~ k e d . ~
mixed m.p. 239.5' (cor.). Anal. Calcd. for (21319.58. Found: N, 19.73. It was also demonstrated that glycine was formed in equimolar quantity with benzaldehyde (Table I). As a preliminary step in ascertaining the effect of substitution of p-hydroxy-a-amino acids, am e t h y l - ~ ~ - s e r i n was e~v~ incubated with pigeon liver extract possessing activity with respect to other P-hydroxy-a-amino acids. No evidence of alanine formation was detected on paper chromatogranis of the enzymatic digest.
The enzyme was purified as described under Experimental. b Benzaldehyde determined directly on the enzymatic digest by measuring the increment in optical density a t 250 mp using a Model DU Beckman spectrophotometer 6 Glycine determined by ninhydrin decarboxylation and colorimetric determination of formaldehyde formed with chromatropic acid.6 d Incubated 30 min. Enzyme preparation more active.
(7) dexlvo-a-SIethylserine has been identified recently as a constituent of t h e antibiotic Amicetin; E. H . Flynn, J. W. Hinman, E. L. Caron and D. 0. Woolf, THISJ O U R N A L , 76, 5867 (1953). ( 8 ) N. K . Sarkar and J. B.Sumner, Enzymologia, 14, 280 (1951).
H10N404: N,
Experimental Enzyme Fractionation.-Rat livers were homogenized in a blendor with two volumes of cold water. The resulting extract was centrifuged a t 42,000 times gravity for two hours. An equal volume of a saturated aqueous solution of ammonium sulfate was added t o the supernatant. The resulting precipitate was taken up in a minimum volume of water TABLE I and dialyzed against water a t 5" until free of ammonia. ENZYMATIC CLEAVAGE OF ISOMERS OF (3-PHESYLSERINEThe dialyzed solution was centrifuged and the precipitate discarded. The supernatant solution was diluted t o a conEnzymatic digests consisted of 2 cc. of 0.1 IM borate centration of 1.5 mg. N/cc. To this solution were added, buffer t o which was added 1 cc. of substrate and 1 cc. of in order, an equal volume of 0.02 M sodium acetate solution purified rat liver enzyme." Incubated a t 37" for 15 min. and a half volume of an aged calcium phosphate gel8 (dry Enzymatic splitting of substrate was linear with respect to weight: 35 mg./cc. of initial suspension). After standing for time over intervals reuorted. one hour the suspension was centrifuged and the enzyme Additions pmoles formed/ eluted from the gel with 0.01 M phosphate buffer pH 7 . 3 . Mg. hr./mg N This enzyme solution was dialyzed against running water a t N/cc. Benzal- G l y umoles Substrate enzyme p H dehydeb cineC 5" for two hours and lyophilized. The activity of the purified rat liver enzyme was 20 p moles of benzaldehyde formed/ 5 threo-8-Phenyl-L-serine 0.27 8 . 3 2.4 hr./mg. r\: when erythfo-P-phenyl-DL-serine was employed 5 threo-P-Phenyl-D-serine .62 8 . 3
