Substrate specificity of human fibroblast stromelysin. Hydrolysis of substance P and its analogues.
To probe the substrate specificity of the human metalloproteinase stromelysin (SLN), we determined values of kc/Km for the SLN-catalyzed hydrolysis of substance P (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-MetNH2; SP; kc/Km = 1790 +/- 140 M-1 s-1), 15 analogues of SP, and 17 other peptides. We found a remarkably narrow substrate specificity for SLN: while SP and its analogues could serve as substrates for SLN (hydrolysis occurred exclusively at the Gln6-Phe7 bond), peptides that were not direct analogues could not (kc/Km less than 3 M-1 s-1). From the study of the SLN-catalyzed hydrolysis of SP and its analogues, the following findings emerged: (1) Decreasing the length of SP results in decreases in kc/Km. (2) Conservative amino acid replacements near the scissle bond of SP decrease kc/Km. (3) The SP analogue in which Gly9 is replaced with sarcosine (N-methylglycine) is not hydrolyzed by SLN (kc/Km less than 3 M-1 s-1). (4) Several SP analogues that are not hydrolyzed by SLN are inhibitors of the enzyme. The complexes formed from interaction of SLN with these peptides have dissociation constants that are similar to the Km value for the complex of SLN and SP. Combined, these results suggest that SLN uses the energy that is available from favorable interactions with its substrate to stabilize catalytic transition states but not the Michaelis complex or other stable-state complexes.
Teahan
J
J
Department of Enzymology, Merck Research Laboratories, Rahway, New Jersey 07065.
Harrison
R
R
Izquierdo
M
M
Stein
R L
RL
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D005347:Fibroblasts / Q000201:enzymology; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D019278:Matrix Metalloproteinase 3; D008666:Metalloendopeptidases / Q000378:metabolism*; D008969:Molecular Sequence Data; D013373:Substance P / Q000031:analogs & derivatives / Q000378:metabolism*; D013379:Substrate Specificity
D013373:Substance P; D008666:Metalloendopeptidases; D019278:Matrix Metalloproteinase 3