Irreversible inhibition of the bacterial cysteine protease-transpeptidase sortase (SrtA) by substrate-derived affinity labels

被引：66

作者：

Scott, CJ ^{[1
]}

McDowell, A ^{[1
]}

Martin, SL ^{[1
]}

Lynas, JF ^{[1
]}

Vandenbroeck, K ^{[1
]}

Walker, B ^{[1
]}

机构：

[1] Queens Univ Belfast, Sch Pharm, Biomol Sci Grp, Belfast BT9 7BL, Antrim, North Ireland

来源：

BIOCHEMICAL JOURNAL | 2002年 / 366卷 / 366期

关键词：

biotinylated probe; peptidyl-chloromethane; peptidyl-diazomethane; recombinant; Western blotting;

D O I：

10.1042/BJ20020602

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

We report on the first synthesis, kinetic evaluation and application of novel substrate-derived inhibitors against the Staphylococcus aurelis cysteine protease-transpeptidase, sortase (staphylococcal surface protein sorting A, SrtA). The peptidyl-diazomethane and peptidyl-chloromethane analogues, Cbz (benzyloxycarbonyl)Leu-Pro-Ala-Thr-CHN2 (I) and Cbz-Leu-Pro-Ala-Thr-CH2Cl (II) respectively were found to act as time-dependent irreversible inhibitors of recombinant sortase (Srt(Deltachi)). The peptidyl-chloromethane analogue (II) was the most powerful with an inhibitor specificity constant (k(i)/k(j)) of 5.3 x 10(4) M-1 . min-1, approx. 2-fold greater than that determined for the peptidyl-diazomethane (I). Additionally, using Western-blot analysis, we have been able to demonstrate that a biotinylated version of the peptidyl-diazomethane analogue, biotin-Ahx (aminohexanoyl)-Leu-Pro-Ala-Thr-CHN2 2 (III), can be used as an affinity label to detect the presence of wild-type SrtA in crude cell lysates prepared from S. aurens.

引用

页码：953 / 958

页数：6

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