Structural investigations on human erythrocyte acylpeptide hydrolase by mass spectrometric procedures.
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 Arg193. The secondary structural organization of the protease domain of the enzyme is consistent with the alpha/beta hydrolase fold.
Scaloni
A
A
Centro Internazionale Servizi di Spettrometria di Massa, C.N.R-Universita' di Napoli Federico II, Naples, Italy. ascaloni@saul.iabbam.na.cnr.it
Ingallinella
P
P
Andolfo
A
A
Jones
W
W
Marino
G
G
Manning
J M
JM
D000478:Alkylation; D000595:Amino Acid Sequence; D000818:Animals; D003545:Cysteine / Q000378:metabolism; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010447:Peptide Hydrolases / Q000032:analysis*; D011499:Protein Processing, Post-Translational; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013552:Swine; D013997:Time Factors; D014357:Trypsin / Q000378:metabolism
D010447:Peptide Hydrolases; C038220:acylaminoacyl-peptidase; D014357:Trypsin; D003545:Cysteine