Chemical modification of tissue inhibitor of metalloproteinases-1 and its inactivation by diethyl pyrocarbonate.
Tissue inhibitor of metalloproteinases-1 (TIMP-1) was treated with a range of chemical modification reagents in order to identify amino acid residues essential for inhibitory activity. Diethyl pyrocarbonate (DEPC) was found to be a potent inactivator at low reagent/TIMP molar concentrations. The extent of modification at 50% inactivation was determined as 1.5 sites/molecule. The DEPC-modified inhibitor did not form stable complexes with stromelysin, but was shown to retain native structure as judged by conformational stability to denaturation by guanidine hydrochloride. Peptide mapping experiments were used to find the sites of DEPC incorporation within the primary structure of TIMP and three residues were identified (His-95, His-144 and His-164). Mutant TIMPs in which histidine residues have been substituted or deleted retain inhibitory activity and were found to be equally as sensitive to DEPC inactivation as the wild-type. No new sites of DEPC modification in the mutant proteins were detected. The possible contribution made by His residues 95, 144 and 164 to the inhibitory activity of TIMP is discussed.
Williamson
R A
RA
Biological Laboratory, University of Kent, Canterbury, UK.
Smith
B J
BJ
Angal
S
S
Freedman
R B
RB
D000595:Amino Acid Sequence; D002250:Carbon Radioisotopes; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology*; D006023:Glycoproteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D006639:Histidine; D006801:Humans; D019278:Matrix Metalloproteinase 3; D008666:Metalloendopeptidases / Q000737:chemistry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000737:chemistry; D019714:Tissue Inhibitor of Metalloproteinases; D014357:Trypsin
D002250:Carbon Radioisotopes; D006023:Glycoproteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D019714:Tissue Inhibitor of Metalloproteinases; D006639:Histidine; D014357:Trypsin; D008666:Metalloendopeptidases; D019278:Matrix Metalloproteinase 3; D004047:Diethyl Pyrocarbonate