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

被引:7
作者
Fernandes, BL
Anéas, MAF
Juliano, L
Palma, MS
Lebrun, I
Portaro, FCV
机构
[1] Univ Sao Paulo, Inst Ciencias Biomed, Dept Microbiol, BR-05508900 Sao Paulo, Brazil
[2] Univ Fed Sao Paulo, Dept Biofis & Biol Mol, Sao Paulo, Brazil
[3] Univ Estadual Sao Paulo, Rio Claro, SP, Brazil
[4] Inst Butantan, Dept Bioquim & Biofis, Sao Paulo, Brazil
关键词
metalloprotease; substrate specificity; quenched fluorescence peptides; Proteus mirabilis;
D O I
10.1590/S0100-879X2000000700006
中图分类号
Q [生物科学];
学科分类号
07 ; 0710 ; 09 ;
摘要
The protease ZapA, secreted by Proteus mirabilis, has been considered to be a virulence factor of this opportunistic bacterium. The control of its expression requires the use of an appropriate methodology, which until now has not been developed. The present study focused on the replacement of azocasein with fluorogenic substrates, and on the definition of enzyme specificity. Eight fluorogenic substrates were tested, and the peptide Abz-Ala-Phe-Arg-Ser-Ala-Ala-Gln-EDDnp was found to be the most convenient for use as an operational 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-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Gln-EDDnp) which was cleaved at a single bond (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 the scissile bonds were also evaluated, and no clear requirement for the amino acid residue at P-1 was found, although the enzyme seems to have a preference for a hydrophobic residue at P-2.
引用
收藏
页码:765 / 770
页数:6
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