The present report presents evidence that the amino acid sequence around the serine of the active site of human tripeptidyl peptidase II is of the subtilisin type. The enzyme from human erythrocytes was covalently labeled at its active site with [3H]diisopropyl fluorophosphate, and the protein was subsequently reduced, alkylated, and digested with trypsin. The labeled tryptic peptides were purified by gel filtration and repeated reversed-phase HPLC, and their aminoterminal sequences were determined. Residue 9 contained the radioactive label and was, therefore, considered to be the active serine residue. The primary structure of the part of the active site (residues 1-10) containing this residue was concluded to be Xaa-Thr-Gln-Leu-Met-Asx-Gly-Thr-Ser-Met. This amino acid sequence is homologous to the sequence surrounding the active serine of the microbial peptidases subtilisin and thermitase. These data demonstrate that human tripeptidyl peptidase II represents a potentially distinct class of human peptidases and raise the question of an evolutionary relationship between the active site of a mammalian peptidase and that of the subtilisin family of serine peptidases.The discovery of an extralysosomal tripeptide-releasing amino-peptidase in rat liver has been reported (1). Its substrate specificity, as studied by use of a series of peptide substrates, is complex (1, 2). The peptidase releases tripeptides with little apparent similarity, although the rate of cleavage varies considerably. The enzyme, currently named tripeptidyl peptidase II (TPP-II) (2, 3), has a high native molecular weight (>106) (1), a subunit molecular weight of 135,000, and a neutral pH optimum (2). The presence ofTPP-II in numerous tissues of the rat (2) and in erythrocytes and liver of several other species (4) has been demonstrated. The human erythrocyte enzyme has been classified as a serine peptidase due to its complete inhibition by diisopropyl fluorophosphate (iPr2P-F) and phenylmethylsulfonyl fluoride (2). However, the enzyme was also inhibited by some of the thiol reagents tested, e.g., N-ethylmaleimide, p-chloromercuribenzoate, and Hg2+. TPP-II could be protected against iPr2P-F, phenylmethylsulfonyl fluoride, or N-ethylmaleimide by the use of a competitive inhibitor (2), suggesting that not only a serine, but also a cysteine residue is of importance for the activity of the enzyme. To obtain an unambiguous classification of TPP-II as a serine peptidase, we considered it necessary to isolate and determine the amino acid sequence of the part of the protein that contains the active serine residue. Information on the primary structure of the active site would also be ofinterest for the investigation of structural relationships between TPP-II and other peptidases.
EXPERIMENTAL PROCEDURES Materials. [1,3-3H]iPr2P-F was purchased from AmershamInternational. Emulsifier scintillator 299 came from Packard. Dithiothreitol and bovine trypsin were from Boehringer Mannheim, and L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin...