Identification and localization of a cysteinyl residue critical for the trypsin-like catalytic activity of the proteasome.
Chemical modification of the proteasome with N-ethylmaleimide (NEM) was performed for the purpose of identifying amino acid residues that play a role in the enzyme's proteolytic function. Modification of the proteasome with NEM specifically and irreversibly suppressed one of the three peptidase activities of the enzyme, viz., the "trypsin-like" activity. Leupeptin, a reversible competitive inhibitor of this activity, protected the activity from NEM inactivation, suggesting that NEM modifies a residue in the leupeptin binding site. Comparisons of enzyme samples labeled with [14C]NEM either in the presence or in the absence of leupeptin allowed the identification of a proteasome subunit containing an NEM-modified, leupeptin-protected cysteinyl residue. The leupeptin protection experiments suggest that residues of this subunit contribute to the active site responsible for the proteasome's trypsin-like activity. This subunit was purified by reverse-phase high-performance liquid chromatography. Peptide mapping and N-terminal amino acid sequencing were employed to acquire information about the primary structure of the subunit, including the sequence surrounding the leupeptin-protected cysteinyl residue. The sequencing data suggest that this proteasome subunit is evolutionarily related to other proteasome subunits that have been sequenced, which show no homology to other known proteases. The assignment of a catalytic function to a member of the proteasome family supports the hypothesis that proteasome subunits represent a structurally and possibly mechanistically novel group of proteases.
Dick
L R
LR
Department of Biochemistry, Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas 75235.
Moomaw
C R
CR
Pramanik
B C
BC
DeMartino
G N
GN
Slaughter
C A
CA
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D002918:Chymotrypsin; D003488:Cyanogen Bromide; D003545:Cysteine; D003546:Cysteine Endopeptidases / Q000097:blood / Q000302:isolation & purification / Q000378:metabolism*; D004912:Erythrocytes / Q000201:enzymology; D005033:Ethylmaleimide / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D007976:Leupeptins / Q000494:pharmacology; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000097:blood / Q000302:isolation & purification / Q000378:metabolism*; D009206:Myocardium / Q000201:enzymology; D010446:Peptide Fragments / Q000302:isolation & purification; D010449:Peptide Mapping; D046988:Proteasome Endopeptidase Complex; D014357:Trypsin / Q000378:metabolism*
D007976:Leupeptins; D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D010446:Peptide Fragments; D002918:Chymotrypsin; D014357:Trypsin; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C032854:leupeptin; D003545:Cysteine; D005033:Ethylmaleimide; D003488:Cyanogen Bromide