ROLE OF THE CONSERVED HISTIDINE AND ASPARTIC-ACID RESIDUES IN ACTIVITY AND STABILIZATION OF HUMAN GELATINASE-B - AN EXAMPLE OF MATRIX METALLOPROTEINASES
T. Pourmotabbed et al., ROLE OF THE CONSERVED HISTIDINE AND ASPARTIC-ACID RESIDUES IN ACTIVITY AND STABILIZATION OF HUMAN GELATINASE-B - AN EXAMPLE OF MATRIX METALLOPROTEINASES, Journal of protein chemistry, 14(7), 1995, pp. 527-535
Gelatinase B (MMP-9), a member of the matrix metalloproteinase family,
is a zinc- and calcium-dependent endopeptidase that is known to play
a role in tumor cell invasion and in destruction of cartilage in arthr
itis, It contains a conserved sequence 00)His-(X)(3)-His-(X)(28)-Asp-A
sp-(X)(2)-(436)Gly, the function of which is under investigation, The
conserved Asp-432 and Asp-433 residues were individually replaced with
Gly; these substitutions reduced the gelatinolytic activity of the en
zyme to 23% and 0%, respectively. Replacing Asp-433 with Glu, however,
decreased the gelatinolytic activity of the enzyme by 93% and proteol
ytic activity of the enzyme for the Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH
2 substrate by 79%, The wild-type and D432G and D433E mutant enzymes h
ad similar K-m values for the synthetic substrate and similar K-i valu
es for the competitive inhibitor, GM6001. The k(cat)/K-m values for D4
32G and D433E mutant enzymes, however, were reduced by a factor of sim
ilar to 4 and their K-a(Ca) values were increased by four- and sixfold
, respectively. The significance of His-400 in the activity of the enz
yme was assessed by replacing this residue with Ala and Phe. Both H400
A and H400F mutants were inactive toward gelatin substrate. These data
demonstrate that Asp-432, Asp-433, and His-400 residues are important
for the activity of gelatinase B, His400 may act as a zinc-binding li
gand similar to the His-197 in interstitial collagenase (MMP-7) and As
p-432 and Asp-433 residues are probably involved in stabilization of t
he active site of the enzyme. The His-400 and Asp-433 residues are con
served in all members of the MMP family. Therefore, our results are re
levant to this group as a whole.