Development of an HPLC assay for Staphylococcus aureus sortase: evidence for the formation of the kinetically competent acyl enzyme intermediate.
Many bacterial surface proteins containing an LPXTG motif are anchored to the cell wall peptidoglycan by catalysis with the thiol transpeptidase sortase. The transpeptidation and hydrolysis reactions of sortase have been proposed to proceed through a common acyl enzyme intermediate. The reactions of Staphylococcus aureus sortase with fluorogenic substrate Abz-LPETG-Dnp in the presence or absence of triglycine were characterized in this study to gain additional insight into the kinetic mechanism of sortase. We report here the development of a reverse-phase HPLC assay to identify and characterize sortase reaction intermediates. The HPLC results provide for the first time clear evidence for the formation of a kinetically competent acyl enzyme intermediate during the overall transpeptidation reaction. The results also suggest that sortase undergoes an unexpected intramolecular acyl transfer reaction in the absence of a nucleophile. The significance of this type of HPLC assay as a tool to study enzyme mechanism is discussed.
Aulabaugh
Ann
A
Department of Chemical and Screening Sciences, Wyeth Research, Collegeville, PA 19426, USA.
Ding
Weidong
W
Kapoor
Bhupesh
B
Tabei
Keiko
K
Alksne
Lefa
L
Dushin
Russ
R
Zatz
Tracy
T
Ellestad
George
G
Huang
Xinyi
X
D019881:Aminoacyltransferases / Q000378:metabolism*; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D017931:DNA Primers; D004795:Enzyme Stability; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013211:Staphylococcus aureus / Q000201:enzymology*
D017931:DNA Primers; D019881:Aminoacyltransferases; D003546:Cysteine Endopeptidases