Staphylococcus aureus sortase transpeptidase SrtA: Insight into the kinetic mechanism and evidence for a reverse protonation catalytic mechanism

Brenda A. Frankel, Ryan G. Kruger, Dana E. Robinson, Neil L. Kelleher, Dewey G. McCafferty*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

111 Scopus citations


The Staphylococcus aureus transpeptidase SrtA catalyzes the covalent attachment of LPXTG-containing virulence and colonization-associated proteins to cell-wall peptidoglycan in Gram-positive bacteria. Recent structural characterizations of staphylococcal SrtA, and related transpeptidases SrtB from S. aureus and Bacillus anthracis, provide many details regarding the active site environment, yet raise questions with regard to the nature of catalysis and active site cysteine thiol activation. Here we re-evaluate the kinetic mechanism of SrtA and shed light on aspects of its catalytic mechanism. Using steady-state, pre-steady-state, bisubstrate kinetic studies, and high-resolution electrospray mass spectrometry, revised steady-state kinetic parameters and a ping-pong hydrolytic shunt kinetic mechanism were determined for recombinant SrtA. The pH dependencies of kinetic parameters kcat/Km and kcat for the substrate Abz-LPETG-Dap(Dnp)-NH2 were bell-shaped with pKa values of 6.3 ± 0.2 and 9.4 ± 0.2 for kcat and 6.2 ± 0.2 and 9.4 ± 0.2 for k cat/Km. Solvent isotope effect (SIE) measurements revealed inverse behavior, with a D2Okcat of 0.89 ± 0.01 and a D2O(kcat/Km) of 0.57 ± 0.03 reflecting an equilibrium SIE. In addition, SIE measurements strongly implicated Cys184 participation in the isotope-sensitive rate-determining chemical step when considered in conjunction with an inverse linear proton inventory for kcat. Last, the pH dependence of SrtA inactivation by iodoacetamide revealed a single ionization for inactivation. These studies collectively provide compelling evidence for a reverse protonation mechanism where a small fraction (ca. 0.06%) of SrtA is competent for catalysis at physiological pH, yet is highly active with an estimated kcat/Km of >10 5 M-1 s-1.

Original languageEnglish (US)
Pages (from-to)11188-11200
Number of pages13
Issue number33
StatePublished - Aug 23 2005

ASJC Scopus subject areas

  • Biochemistry


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