TY - JOUR
T1 - Structural investigations on human erythrocyte acylpeptide hydrolase by mass spectrometric procedures
AU - Scaloni, Andrea
AU - Ingallinella, Paolo
AU - Andolfo, Annapaola
AU - Jones, Wanda
AU - Marino, Gennaro
AU - Manning, James M.
PY - 1999
Y1 - 1999
N2 - The complete primary structure of human erythrocyte acylpeptide hydrolase has been determined by using a combination of different mass spectrometric procedures and sequencing techniques. These data allowed us to correct the incomplete nucleotide sequence of the DNF15S2 locus on the short arm of human 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 experiments on the native enzyme demonstrated that all 17 cysteine residues present in the polypeptide chain are in reduced form. Multiple sequence alignment did not reveal striking similarity with proteases of known tertiary structure 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 in a completely active nicked form. The site of tryptic hydrolysis was identified as Arg 193. The secondary structural organization of the protease domain of the enzyme is consistent with the α/β hydrolase fold.
AB - The complete primary structure of human erythrocyte acylpeptide hydrolase has been determined by using a combination of different mass spectrometric procedures and sequencing techniques. These data allowed us to correct the incomplete nucleotide sequence of the DNF15S2 locus on the short arm of human 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 experiments on the native enzyme demonstrated that all 17 cysteine residues present in the polypeptide chain are in reduced form. Multiple sequence alignment did not reveal striking similarity with proteases of known tertiary structure 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 in a completely active nicked form. The site of tryptic hydrolysis was identified as Arg 193. The secondary structural organization of the protease domain of the enzyme is consistent with the α/β hydrolase fold.
KW - Acylpeptide hydrolase
KW - Cysteine residues
KW - Limited proteolysis
KW - Post-translational modifications
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U2 - 10.1023/A:1021047730831
DO - 10.1023/A:1021047730831
M3 - Article
C2 - 10395453
VL - 18
SP - 349
EP - 360
JO - Journal of Protein Chemistry
JF - Journal of Protein Chemistry
SN - 0277-8033
IS - 3
ER -