Staphylococcus aureus Sortase Transpeptidase SrtA: Insight into the Kinetic Mechanism and Evidence for a Reverse Protonation Catalytic Mechanism

详细信息    查看全文

• 作者：Brenda A. Frankel ; Ryan G. Kruger ; Dana E. Robinson ; Neil L. Kelleher ; and Dewey G. McCafferty
• 刊名：Biochemistry
• 出版年：2005
• 出版时间：August 23, 2005
• 年：2005
• 卷：44
• 期：33
• 页码：11188 - 11200
• 全文大小：312K
• 年卷期：v.44,no.33(August 23, 2005)
• ISSN：1520-4995

文摘

The Staphylococcus aureus transpeptidase SrtA catalyzes the covalent attachment of LPXTG-containing virulence and colonization-associated proteins to cell-wall peptidoglycan in Gram-positivebacteria. Recent structural characterizations of staphylococcal SrtA, and related transpeptidases SrtB fromS. aureus and Bacillus anthracis, provide many details regarding the active site environment, yet raisequestions 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 determinedfor recombinant SrtA. The pH dependencies of kinetic parameters k_cat/K_m and k_cat for the substrate Abz-LPETG-Dap(Dnp)-NH₂ 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, witha ^D₂Ok_cat of 0.89 ± 0.01 and a ^D₂O(k_cat/K_m) of 0.57 ± 0.03 reflecting an equilibrium SIE. In addition, SIEmeasurements strongly implicated Cys184 participation in the isotope-sensitive rate-determining chemicalstep when considered in conjunction with an inverse linear proton inventory for k_cat. Last, the pH dependenceof SrtA inactivation by iodoacetamide revealed a single ionization for inactivation. These studies collectivelyprovide compelling evidence for a reverse protonation mechanism where a small fraction (ca. 0.06%) ofSrtA is competent for catalysis at physiological pH, yet is highly active with an estimated k_cat/K_m of >10⁵M^-1 s^-1.