i3-Chloroamino acids have been shown recently to bind efficiently to transaminases and decarboxylases and to undergo #3-elimination reactions in situ (7,8). In some cases the enzyme catalyzing the (3-elimination reaction is irreversibly inactivated presumably by reaction of the enzyme-bound aminoacrylic acid with some functional group of the protein (8). Accordingly, it has occurred to us that (3-chloro-D-alanine might inhibit irreversibly the pyridoxal phosphate enzymes responsible for the formation of D-alanine in bacterial cells and thereby preclude the synthesis of a cell-wall constituent necessary for bacterial growth. MATERIALSThe D-and -isomers of fl-chloroalanine were purchased from Cyclo as the hydrochloride salts. Elemental analysis of (3-chloro-D-alanine gave: C, 22. METHODSConditions for Growth of Bacteria. For periodic measurement of the effect of (3-chloro-D-alanine on the growth of pneumococcus, portions of cultures in the logarithmic phase of growth were added under sterile conditions to 9 volumes of a minimal medium devoid of alanine (9) in 18 X 150-mm tubes. The compounds to be tested (the i-and D-isomers of 0-chloroalanine, D-and L-alanine, and D-alanyl-D-alanine) were then added, and incubations were carried out at 37°. In these experiments bacterial growth was measured by nephelometry on a Coleman instrument periodically for 3-6 hr.For determination of enzyme activities and of free intracellular alanine, cultures of either E. coli or B. subtilis were grown aerobically at 370 in 25-ml Erlenmeyer flasks in yeast extract-supplemented medium [0.5% dialyzed Difco yeast extract added to the minimal medium of Frantz (10)] and nutrient broth, respectively. The cultures were treated with 3-4 mM fl-chloro-D-alanine while in the logarithmic phase of growth, and incubated for an additional 3-4 hr. The cells were collected by centrifugation at 23,000 X g at 40 for 10 min. After being washed in 5-10 ml of 0.85% NaCl, they were resuspended in 10 ml 0.1 M potassium phosphate, pH 7.0. The cells were ruptured by sonication for 1-2 min at 00 in a Branson 750-watt sonicator at a setting of 3; the sonicate was then centrifuged for 10 min at 27,000 X g and 4°. The concentration of protein in the extract was estimated by the absorbancies at 280 and 260 nm (11).Determination' of the Amounts and Configurations ofChloroalanine and Alanine. The amounts of fl-chloroalanine and alanine in the culture supernatants and extracts of E. coli and B. subtilis were determined by amino-acid analysis (12, 13). Under our experimental conditions, (3-chloroalanine eluted at 58 ml and alanine at 122 ml from the 0.9 X 50-cm

An 87% identity has been found between the reported cDNA sequence that encodes acylpeptide hydrolase (EC 3.4.19.1) [Mitta, M., Asada, K., Uchimura, Y., Kimizuka, F., Kato, I., Sakiyama, F. & Tsunasawa, S. (1989) J. Biochem. 106, 548-551] and a cDNA transcribed from a locus (DNFISS2) on the short arm ofhuman chromosome 3, reported by Naylor et al. [Naylor, S. L., Marshall, A., Hensel, C., Martinez, P. F., Holley, B. & Sakaguchi, A. Y. (1989) Genomics 4,[355][356][357][358][359][360][361]; the DNF15S2 locus suffers deletions in small cell lung carcinoma associated with a reduction or loss of acylase activity (EC 3.5.1.14). Acylpeptide hydrolase catalyzes the hydrolysis of the terminal acetylated amino acid preferentially from small acetylated peptides. The acetylamino acid formed by acylpeptide hydrolase is further processed to acetate and a free amino acid by an acylase. The substrates for the acylpeptide hydrolase and the acylase behave in a reciprocal manner since acylpeptide hydrolase binds but does not process acetylamino acids and the acylase binds acetylpeptides but does not hydrolyze them; however, the two enzymes share the same specificity for the acyl group. These findings indicate some common functional features in the protein structures of these two enzymes. Since the gene coding for acylpeptide hydrolase is within the same region of human chromosome 3 (3p2l) that codes for the acylase and deletions at this locus are also associated with a decrease in acylase activity, there is a close genetic relationship between the two enzymes. There could also be a relationship between the expression of these two enzymes and acetylated peptide growth factors in some carcinomas.The various types of exopeptidases that act on the free NH2-terminal residues of polypeptides have been described in detail (1). The properties of a purified enzyme that cleaves an acetylated terminal amino acid from acetylated peptides (N-acylaminoacylpeptide hydrolase, EC 3.4.19.1, referred to here as acylpeptide hydrolase) have also been reported (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). For example, this enzyme catalyzes the hydrolysis of acetyltrialanine (Ac-Ala3-OH) to acetylalanine (Ac-Ala-OH) and dialanine (Ala2-OH). We reported that the rates of hydrolysis of different blocked peptide substrates varied considerably, depending on the nature of the first and second amino acids. Thus, there was a preference for Ac-Ala-, Ac-Met-, and Ac-Ser-at the blocked terminus (4). Comparison ofthis specificity with the sequences ofabout 100 known proteins acetylated at their NH2-terminal residues indicated that most of them began with Ac-Ala-, Ac-Met-, or Ac-Ser-(13). Furthermore, in these blocked proteins there was a preponderance of charged amino acid residues at the second position. Hence, the characteristics of the terminal sequence of these blocked proteins appear to resemble the substrate specificity ofthe acylpeptide hydrolase. The enzyme displays a broad spectrum with respect to the blocking group, since acetyl, chloroacetyl, formyl,...

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