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

Publication ,  Journal Article
Frankel, BA; Kruger, RG; Robinson, DE; Kelleher, NL; McCafferty, DG
Published in: Biochemistry
August 2005

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 k(cat)/K(m) and k(cat) for the substrate Abz-LPETG-Dap(Dnp)-NH(2) were bell-shaped with pK(a) values of 6.3 +/- 0.2 and 9.4 +/- 0.2 for k(cat) and 6.2 +/- 0.2 and 9.4 +/- 0.2 for k(cat)/K(m). Solvent isotope effect (SIE) measurements revealed inverse behavior, with a (D)2(O)k(cat) of 0.89 +/- 0.01 and a (D)2(O)(k(cat)/K(m)) 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 k(cat). 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 k(cat)/K(m) of >10(5) M(-)(1) s(-)(1).

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Published In

Biochemistry

DOI

EISSN

1520-4995

ISSN

0006-2960

Publication Date

August 2005

Volume

44

Issue

33

Start / End Page

11188 / 11200

Related Subject Headings

  • Staphylococcus aureus
  • Spectrometry, Mass, Electrospray Ionization
  • Kinetics
  • Hydrogen-Ion Concentration
  • Enzyme Activation
  • Cysteine Endopeptidases
  • Cysteine
  • Cell Wall
  • Catalysis
  • Biochemistry & Molecular Biology
 

Citation

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Frankel, B. A., Kruger, R. G., Robinson, D. E., Kelleher, N. L., & McCafferty, D. G. (2005). Staphylococcus aureus sortase transpeptidase SrtA: insight into the kinetic mechanism and evidence for a reverse protonation catalytic mechanism. Biochemistry, 44(33), 11188–11200. https://doi.org/10.1021/bi050141j
Frankel, Brenda A., Ryan G. Kruger, Dana E. Robinson, Neil L. Kelleher, and Dewey G. McCafferty. “Staphylococcus aureus sortase transpeptidase SrtA: insight into the kinetic mechanism and evidence for a reverse protonation catalytic mechanism.Biochemistry 44, no. 33 (August 2005): 11188–200. https://doi.org/10.1021/bi050141j.
Frankel BA, Kruger RG, Robinson DE, Kelleher NL, McCafferty DG. Staphylococcus aureus sortase transpeptidase SrtA: insight into the kinetic mechanism and evidence for a reverse protonation catalytic mechanism. Biochemistry. 2005 Aug;44(33):11188–200.
Frankel, Brenda A., et al. “Staphylococcus aureus sortase transpeptidase SrtA: insight into the kinetic mechanism and evidence for a reverse protonation catalytic mechanism.Biochemistry, vol. 44, no. 33, Aug. 2005, pp. 11188–200. Epmc, doi:10.1021/bi050141j.
Frankel BA, Kruger RG, Robinson DE, Kelleher NL, McCafferty DG. Staphylococcus aureus sortase transpeptidase SrtA: insight into the kinetic mechanism and evidence for a reverse protonation catalytic mechanism. Biochemistry. 2005 Aug;44(33):11188–11200.
Journal cover image

Published In

Biochemistry

DOI

EISSN

1520-4995

ISSN

0006-2960

Publication Date

August 2005

Volume

44

Issue

33

Start / End Page

11188 / 11200

Related Subject Headings

  • Staphylococcus aureus
  • Spectrometry, Mass, Electrospray Ionization
  • Kinetics
  • Hydrogen-Ion Concentration
  • Enzyme Activation
  • Cysteine Endopeptidases
  • Cysteine
  • Cell Wall
  • Catalysis
  • Biochemistry & Molecular Biology