Irreversible inhibition of the bacterial cysteine protease-transpeptidase sortase (SrtA) by substrate-derived affinity labels.
We report on the first synthesis, kinetic evaluation and application of novel substrate-derived inhibitors against the Staphylococcus aureus cysteine protease-transpeptidase, sortase (staphylococcal surface protein sorting A, SrtA). The peptidyl-diazomethane and peptidyl-chloromethane analogues, Cbz (benzyloxycarbonyl)-Leu-Pro-Ala-Thr-CHN(2) (I) and Cbz-Leu-Pro-Ala-Thr-CH(2)Cl (II) respectively were found to act as time-dependent irreversible inhibitors of recombinant sortase (SrtA(DeltaN)). The peptidyl-chloromethane analogue (II) was the most powerful with an inhibitor specificity constant (k(i)/K(i)) of 5.3x10(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-CHN(2) (III), can be used as an affinity label to detect the presence of wild-type SrtA in crude cell lysates prepared from S. aureus.
Scott
Christopher J
CJ
Biomolecular Sciences Group, School of Pharmacy, The Queen's University of Belfast, Northern Ireland, UK.
McDowell
Andrew
A
Martin
S Lorraine
SL
Lynas
John F
JF
Vandenbroeck
Koen
K
Walker
Brian
B
D019881:Aminoacyltransferases / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D001426:Bacterial Proteins; D019921:Biotinylation; D015153:Blotting, Western; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000737:chemistry*; D003978:Diazomethane / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007700:Kinetics; D008737:Methyl Chloride / Q000737:chemistry; D008956:Models, Chemical; D010458:Peptidyl Transferases / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013211:Staphylococcus aureus / Q000201:enzymology
D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D003978:Diazomethane; D008737:Methyl Chloride; D019881:Aminoacyltransferases; C120542:sortase A; D010458:Peptidyl Transferases; D003546:Cysteine Endopeptidases