Inhibition of human type IV collagenase by a highly conserved peptide sequence derived from its prosegment.
The proenzyme fragment of the 72 kDa type IV collagenase contains a conserved amino acid sequence, MRKPRCGN(V)PDV, that is shared with other members of the matrix metalloproteinase family, such as interstitial collagenase and stromelysin. This sequence is lost upon the autocatalytic removal of the 80-84 amino acids from the amino terminus of these proenzymes following enzyme activation. The loss of this profragment converts the latent proenzyme species into a stable active enzyme species. In the present study, we demonstrate that this conserved prosegment sequence is an inhibitor of these enzymes and plays a critical role in maintenance of the latent state of the matrix metalloproteinases. Peptides containing the conserved sequence, MRKPRCGNPDV, were capable of inhibiting activated enzyme. Free cysteine was also an effective inhibitor, whereas reduced glutathione was a less effective inhibitor. Oxidized glutathione was not inhibitory. The 72 kDa type IV collagenase holoproenzyme preparations did not contain a free cysteinyl side chain that reacted with the sulfhydryl substitution reagent 5,5'-dithiobis(2-nitrobenzoic acid) (Ellman's reagent). However, addition of ethylenediaminetetraacetic acid to the reaction mixture to generate the apoenzyme form resulted in the detection of titrable sulfhydryl side chains. Based on these data, we postulate that in the latent enzyme state the conserved profragment sequence interacts with the metal atom at the active site through a sulfhydryl-metal atom coordination that is further stabilized by the amino acyl residues surrounding the essential 73Cys residue. Disturbance of this interaction results in enzyme activation.(ABSTRACT TRUNCATED AT 250 WORDS)
Stetler-Stevenson
W G
WG
Tumor Invasion and Metastasis Section, Laboratory of Pathology, National Cancer Institute, Bethesda, Maryland 20892.
Talano
J A
JA
Gallagher
M E
ME
Krutzsch
H C
HC
Liotta
L A
LA
D000595:Amino Acid Sequence; D000818:Animals; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000494:pharmacology*; D006801:Humans; D003012:Microbial Collagenase / Q000037:antagonists & inhibitors* / Q000235:genetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000494:pharmacology*
D004792:Enzyme Precursors; D010446:Peptide Fragments; D003012:Microbial Collagenase