TABLE 2.
Origin | Form of MurD | Vmax (nmol/min · mg)a |
Km(app) (μM)a
|
Vmax/Km(app, UDPNacMur-l-Ala) | Relative efficiency | ||
---|---|---|---|---|---|---|---|
UDPNacMur-l-Alab | ATP | d-Glutamatec | |||||
S. aureus | MBP-MurD fusion protein | 31,787 ± 3,668 | 245 ± 29 | 78 ± 9 | 660 ± 51 | 12,974 | 0.14 |
Regenerated MurD | 32,118 ± 3,636 | 290 ± 34 | 84 ± 10 | 534 ± 25 | 11,075 | 0.12 | |
E. faecalis | MBP-MurD fusion protein | 32,857 ± 3,647 | 38 ± 4 | 44 ± 5 | 121 ± 10 | 86,466 | 0.90 |
Regenerated MurD | 29,823 ± 5,934 | 36 ± 7 | 47 ± 4 | 118 ± 14 | 82,842 | 0.87 | |
E. coli | MBP-MurD fusion protein | 5,965 ± 297 | 5 ± 2 | 154 ± 23 | 27 ± 12 | 85,214 | 0.89 |
Regenerated MurD | 4,783 ± 423 | 7 ± 3 | 135 ± 33 | 42 ± 11 | 95,660 | 1.00 | |
H. influenzae | MBP-MurD fusion protein | NDd | ND | ND | ND | ND | ND |
Regenerated MurD | 13,111 ± 2,199 | 8 ± 4 | 102 ± 6 | 169 ± 20 | 163,888 | 1.71 |
Gram-positive MurD activity was determined with the coupled PK-LDH assay (up to 10 min).
Gram-negative MurD (1- to 2-min assay) was assayed by HPLC or coupled enzyme assay.
All assays were done in duplicate, and a minimal of three independent experiments were conducted for each kinetic determination.
Reaction rates within the linear region of product formation were used for parameter analysis.
0 to 2 mM UDP-N-acetylmuramyl-l-alanine (UDPNacMur-l-Ala) was used for gram-positive MurD assay; 0 to 200 μM was used for gram-negative MurD assay.
The ranges of ATP and d-glutamate used in gram-negative MurD assays were from 0 to 1 mM and from 0 to 2 mM, respectively.
The ranges of ATP and d-glutamate used in gram-positive MurD assays were from 0 to 1 mM and from 0 to 5 mM, respectively.
ND, not determined.