Development of an operational substrate for ZapA, a metalloprotease secreted by the bacterium Proteus mirabilis

The protease ZapA, secreted by Proteus mirabilis, has been considered to be a virulence factor of this opportunistic bacterium. The control of its exp ression requires the use of an appropriate methodology, which until now has not been developed. The present study focused on the replacement of azocas ein with fluorogenic substrates, and on the definition of enzyme specificit y. Eight fluorogenic substrates were tested, and the peptide Abz-Ala-Phe-Ar g-Ser-Ala-Ala-Gln-EDDnp was found to be the most convenient for use as an o perational substrate for ZapA. A single peptide bond (Arg-Ser) was cleaved with a K-m of 4.6 mu M, a k(cat) of 1.73 s(-1), and a catalytic efficiency of 376 (mM s)(-1). Another good substrate for ZapA was peptide 6 (Abz-Arg-P ro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Gln-EDDnp) which was cleaved at a single bon d (Phe-Ser) with a K-m of 13.6 mu M, a k(cat) of 3.96 s(-1) and a catalytic efficiency of 291 (mM s)(-1). The properties of the amino acids flanking t he scissile bonds were also evaluated, and no clear requirement for the ami no acid residue at P-1 was found, although the enzyme seems to have a prefe rence for a hydrophobic residue at P-2.