Neisseria gonorrhoeae Penicillin-Binding Protein 3 Exhibits Exceptionally High Carboxypeptidase and -Lactam Bi
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- 作者：Miglena E. Stefanova ; Joshua Tomberg ; Melanie Olesky ; Joachim-Volker Hö ; ltje ; William G. Gutheil ; and Robert A. Nicholas
- 刊名：Biochemistry
- 出版年：2003
- 出版时间：December 16, 2003
- 年：2003
- 卷：42
- 期：49
- 页码：14614 - 14625
- 全文大小：440K
- 年卷期：v.42,no.49(December 16, 2003)
- ISSN：1520-4995
文摘
A soluble form of penicillin-binding protein 3 (PBP 3) from Neisseria gonorrhoeae wasexpressed and purified from Escherichia coli and characterized for its interaction with 
-lactam antibiotics,its catalytic properties with peptide and peptidoglycan substrates, and its role in cell viability andmorphology. PBP 3 had an unusually high k2/K' value relative to other PBPs for acylation with penicillin(7.7 × 105 M-1 s-1) at pH 8.5 at 25 
C and hydrolyzed bound antibiotic very slowly (k3 < 4.6 × 10-5s-1, t1/2 > 230 min). PBP 3 also demonstrated exceptionally high carboxypeptidase activity with a kcat of580 s-1 and a kcat/Km of 1.8 × 105 M-1 s-1 with the substrate N
-Boc-N
-Cbz-L-Lys-D-Ala-D-Ala. This isthe highest kcat value yet reported for a PBP or other serine peptidases. Activity against a ~D-Ala-D-Lacpeptide substrate was ~2-fold lower than against the analogous ~D-Ala-D-Ala peptide substrate, indicatingthat deacylation is rate determining for both amide and ester hydrolysis. The pH dependence profiles ofboth carboxypeptidase activity and -lactam acylation were bell-shaped with maximal activity at pH 8.0-8.5. PBP 3 displayed weak transpeptidase activity in a model transpeptidase reaction but was active as anendopeptidase, cleaving dimeric peptide cross-links. Deletion of PBP 3 alone had little effect on viability,growth rate, and morphology of N. gonorrhoeae, although deletion of both PBP 3 and PBP 4, the otherlow-molecular-mass PBP in N. gonorrhoeae, resulted in a decreased growth rate and marked morphologicalabnormalities.
-lactam antibiotics,its catalytic properties with peptide and peptidoglycan substrates, and its role in cell viability andmorphology. PBP 3 had an unusually high k2/K' value relative to other PBPs for acylation with penicillin(7.7 × 105 M-1 s-1) at pH 8.5 at 25 C and hydrolyzed bound antibiotic very slowly (k3 < 4.6 × 10-5s-1, t1/2 > 230 min). PBP 3 also demonstrated exceptionally high carboxypeptidase activity with a kcat of580 s-1 and a kcat/Km of 1.8 × 105 M-1 s-1 with the substrate N-Boc-N-Cbz-L-Lys-D-Ala-D-Ala. This isthe highest kcat value yet reported for a PBP or other serine peptidases. Activity against a ~D-Ala-D-Lacpeptide substrate was ~2-fold lower than against the analogous ~D-Ala-D-Ala peptide substrate, indicatingthat deacylation is rate determining for both amide and ester hydrolysis. The pH dependence profiles ofboth carboxypeptidase activity and -lactam acylation were bell-shaped with maximal activity at pH 8.0-8.5. PBP 3 displayed weak transpeptidase activity in a model transpeptidase reaction but was active as anendopeptidase, cleaving dimeric peptide cross-links. Deletion of PBP 3 alone had little effect on viability,growth rate, and morphology of N. gonorrhoeae, although deletion of both PBP 3 and PBP 4, the otherlow-molecular-mass PBP in N. gonorrhoeae, resulted in a decreased growth rate and marked morphologicalabnormalities.