Membrane type-1 matrix metalloprotease and stromelysin-3 cleave more efficiently synthetic substrates containing unusual amino acids in their P1' positions.
The influence of the substrate P1' position on the specificity of two zinc matrix metalloproteases, membrane type-1 matrix metalloprotease (MT1-MMP) and stromelysin-3 (ST3), was evaluated by synthesizing a series of fluorogenic substrates of general formula dansyl-Pro-Leu-Ala-Xaa-Trp-Ala-Arg-NH2, where Xaa in the P1' position represents unusual amino acids containing either long arylalkyl or alkyl side chains. Our data demonstrate that both MT1-MMP and ST3 cleave substrates containing in their P1' position unusual amino acids with extremely long side chains more efficiently than the corresponding substrates with natural phenylalanine or leucine amino acids. In this series of substrates, the replacement of leucine by S-para-methoxybenzyl cysteine increased the kcat/Km ratio by a factor of 37 for MT1-MMP and 9 for ST3. The substrate with a S-para-methoxybenzyl cysteine residue in the P1' position displayed a kcat/Km value of 1.59 10(6) M-1 s-1 and 1.67 10(4) M-1 s-1, when assayed with MT1-MMP and ST3, respectively. This substrate is thus one of the most rapidly hydrolyzed substrates so far reported for matrixins, and is the first synthetic peptide efficiently cleaved by ST3. These unexpected results for these two matrixins suggest that extracellular proteins may be cleaved by matrixins at sites containing amino acids with unusual long side chains, like those generated in vivo by some post-translational modifications.
Mucha
A
A
CEA, Département d'Ingénierie et d'Etudes des Protéines, CE-Saclay, 91191 Gif/Yvette Cedex, France.
Cuniasse
P
P
Kannan
R
R
Beau
F
F
Yiotakis
A
A
Basset
P
P
Dive
V
V
D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D002863:Chromogenic Compounds / Q000378:metabolism; D003619:Dansyl Compounds / Q000378:metabolism; D053507:Matrix Metalloproteinase 11; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases / Q000378:metabolism*; D008667:Metalloproteins / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism*; D013379:Substrate Specificity; D015032:Zinc
D000596:Amino Acids; D002863:Chromogenic Compounds; D003619:Dansyl Compounds; D008667:Metalloproteins; D009842:Oligopeptides; D053507:Matrix Metalloproteinase 11; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases; D015032:Zinc