Cleavage of bovine collagen I by neutrophil collagenase MMP-8. Effect of pH on the catalytic properties as compared to synthetic substrates.
The enzymatic processing of bovine collagen I by neutrophil collagenase (MMP-8) has been monitored at 37 degrees C, envisaging the occurrence of multiple intermediate steps, following the initial cleavage, which leads to the formation of (1/4) and (3/4) fragments. Further, the first cleavage event has been investigated at 37 degrees C as a function of pH, and catalytic parameters have been obtained through a global analysis of steady-state kinetic data, such as to get an overall consistent picture of k(cat)/K(m), k(cat), and K(m). These data have been compared with those obtained from the catalysis by MMP-8 of two synthetic fluorogenic substrates under the same experimental conditions. The overall behavior can be accounted for by the existence of five protonating groups, which vary to a different extent their pK(a) values for the three substrates investigated. The main observation concerns the fact the two of these residues, which play a relevant role in the enzymatic activity of MMP-8, are relatively far from the primary recognition site, and they are coming into action only for large macromolecular substrates, such as bovine collagen I. This finding opens the question of appropriate testing for inhibitors of the enzymatic action of MMP-8, which must take into account, and also of these relevant interactions occurring only with natural substrates.
Marini
S
S
Department of Experimental Medicine and Biochemical Sciences, University of Roma Tor Vergata, Via di Tor Vergata 135, I-00133 Roma, Italy.
Fasciglione
G F
GF
de Sanctis
G
G
D'Alessio
S
S
Politi
V
V
Coletta
M
M
D000818:Animals; D002384:Catalysis; D002417:Cattle; D003094:Collagen / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D020784:Matrix Metalloproteinase 8 / Q000378:metabolism*; D009504:Neutrophils / Q000201:enzymology*; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity
D011994:Recombinant Proteins; D003094:Collagen; D020784:Matrix Metalloproteinase 8