Structural investigations on human erythrocyte acylpeptide hydrolase by mass spectrometric procedures

The complete primary structure of human erythrocyte acylpeptide hydrolase h as been determined by using a combination of different mass spectrometric p rocedures and sequencing techniques. These data allowed us to correct the i ncomplete nucleotide sequence of the DNF15S2 locus on the short arm of huma n chromosome 3 at region 21, coding for the enzyme. The protein consists of 732 amino acid residues and is acetylated at the N-terminus. Alkylation ex periments on the native enzyme demonstrated that all 17 cysteine residues p resent in the polypeptide chain are in reduced form. Multiple sequence alig nment did not reveal striking similarity with proteases of known tertiary s tructure with the exception of members of the serine oligopeptidase family. Limited proteolysis experiments generated a C-terminal portion, containing all the catalytic triad elements responsible for proteolytic activity, and an N-terminal domain of unknown function, both still strongly associated i n a completely active nicked form. The site of tryptic hydrolysis was ident ified as Arg193. The secondary structural organization of the protease doma in of the enzyme is consistent with the alpha/beta hydrolase fold.