determined by a least-squares fit of the initial rate data. One unit
of incubation, the reaction was initiated by the addition of 5 mL
of E. coli DHQS (35 nM).
(1 U) of enzyme activity is defined as the production of 1 mmol
◦
of shikimate product per minute at 25 C for E. coli DHQS and
Steady state kinetics for A. chinensis DHQS with (3R)-3-
fluoroDAH7P. The standard assay reaction mixture contained
(3R)-3-fluoroDAH7P (4 to 160 mM) at pH 6.8 at 25 ◦C. The
mixture was preincubated at 25 ◦C for 1 min followed by addition
of 20 mL of E. coli DHQase (10 mg mL-1). After a further minute
of incubation, the reaction was initiated by the addition of 4 mL
of A. chinensis DHQS (0.13 mM).
◦
A. chinensis DHQS, and at 60 C for P. furiosus DHQS. Specific
activity is defined as the production of 1 mmol of dehydroshikimate
product per minute at 25 ◦C per mg of protein (U mg-1). Km and
kcat values were determined by fitting the data to the Michaelis–
Menten equation using GraFit 5 (Erithacus Software Limited,
2006). The total volume for all assays was 1000 mL. Errors between
measurements were no more than 10%.
Steady state kinetics for P. furiosus DHQS with (3R)-3-
fluoroDAH7P. The standard assay reaction mixture contained
(3R)-3-fluoroDAH7P (4 to 241 mM) at pH 6.7 at 60 ◦C. The
mixture was preincubated at 60 ◦C for 5 min followed by addition
of 20 mL of P. furiosus DHQase (4.1 mg mL-1). After a further
minute of incubation, the reaction was initiated by the addition of
10 mL of P. furiosus DHQS (56 nM).
Steady state kinetics for E. coli DHQS with DAH7P. The
standard assay reaction mixtures contained DAH7P (0.5 to 42 mM)
at pH 6.8. The mixture was preincubated at 25 ◦C for 1 min
followed by addition of 20 mL of E. coli DHQase (10 mg mL-1).
After a further minute of incubation, the reaction was initiated by
the addition of 20 mL of E. coli DHQS (7 nM).
Steady state kinetics for A. chinensis DHQS with DAH7P. The
standard assay reaction mixture contained DAH7P (5 to 500 mM)
at pH 6.8. The mixture was preincubated at 25 ◦C for 1 min
followed by addition of 20 mL of E. coli DHQase (10 mg mL-1).
After a further minute of incubation, the reaction was initiated by
the addition of 5 mL of A. chinensis DHQS (86 nM).
Notes and references
1 B. Ganem, Tetrahedron, 1978, 34, 3353–3383.
2 J. R. Knowles, Aldrichimica Acta, 1989, 22, 59–66.
3 A. Gunel-Ozcan, K. A. Brown, A. G. Allen and D. J. Maskell, Microbial
Pathogen, 1997, 17, 169–174.
4 S. L. Bender, S. Mehdi and J. R. Knowles, Biochemistry, 1989, 28, 7555–
7560; S. L. Bender, T. S. Widlanski and J. R. Knowles, Biochemistry,
1989, 28, 7560–7572; J. W. Frost and J. R. Knowles, Biochemistry,
1984, 23, 4465–4469; T. S. Widlanski, S. L. Bender and J. R. Knowles,
Biochemistry, 1989, 28, 7572–7582; T. S. Widlanski, S. L. Bender and
J. R. Knowles, J. Am. Chem. Soc., 1987, 109, 1873–1875.
5 J. W. Frost, J. L. Bender, J. T. Kadonaga and J. R. Knowles,
Biochemistry, 1984, 23, 4470–4475.
Steady state kinetics for P. furiosus DHQS with DAH7P. The
standard assay reaction mixture contained DAH7P (1 to 70 mM) at
pH 6.7 at 60 ◦C. The mixture was preincubated at 60 ◦C for 5 min
followed by addition of 20 mL of P. furiosus DHQase (4.1 mg mL-1).
After a further minute of incubation, the reaction was initiated by
the addition of 10 mL of P. furiosus DHQS (56 nM).
6 T. S. Widlanski, S. L. Bender and J. R. Knowles, J. Am. Chem. Soc.,
1989, 111, 2299–2300.
7 P. A. Bartlett and K. Satake, J. Am. Chem. Soc., 1988, 110, 1628–1630.
8 S. L. Rotenberg and D. B. Sprinson, Proc. Natl. Acad. Sci. U. S. A., 1970,
67, 1669–1672; S. L. Rotenberg and D. B. Sprinson, J. Biol. Chem., 1978,
253, 2210–2215; M. J. Turner, B. W. Smith and E. Haslam, J. Chem.
Soc., Perkin Trans. 1, 1975, 52–55.
9 P. R. Srinivasan, J. Rothschild and D. B. Sprinson, J. Biol. Chem., 1963,
238, 3176–3182.
Steady state kinetics for E. coli DHQS with (3S) - 3-
fluoroDAH7P. The standard assay reaction mixture contained
(3S)-3-fluoroDAH7P (5 to 240 mM) at pH 6.8 at 25 ◦C. The
mixture was preincubated at 25 ◦C for 1 min followed by addition
of 20 mL of E. coli DHQase (10 mg mL-1). After a further minute
of incubation, the reaction was initiated by the addition of 20 mL
of E. coli DHQS (7 nM).
10 P. A. Bartlett, K. L. McLaren and M. A. Marx, J. Org. Chem., 1994,
59, 2082–2085.
11 E. P. Carpenter, A. R. Hawkins, J. W. Frost and K. A. Brown, Nature,
1998, 394, 299–302.
Steady state kinetics for A. chinensis DHQS with (3S)-3-
fluoroDAH7P. The standard assay reaction mixture contained
(3S)-3-fluoroDAH7P (8 to 93 mM) at pH 6.8 at 25 ◦C. The mixture
was preincubated at 25 ◦C for 1 min followed by addition of
20 mL of E. coli DHQase (10 mg mL-1). After a further minute of
incubation, the reaction was initiated by the addition of 10 mL of
A. chinensis DHQS (86 nM).
12 E. J. Parker, J. R. Coggins and C. Abell, J. Org. Chem., 1997, 62, 8582–
8585.
13 P. F. Pilch and R. L. Somerville, Biochemistry, 1976, 15, 5315–5320.
14 I. P. Street, K. Rupitz and S. G. Withers, Biochemistry, 1989, 28, 1581–
1587.
15 N. T. Anh and O. Eisenstein, Nouveau Journal de Chimie, 1977, 1, 61–70;
A. S. Cieplak, Chem. Rev. (Washington, D. C.), 1999, 99, 1265–1336;
A. S. Cieplak, B. D. Tait and C. R. Johnson, J. Am. Chem. Soc., 1989,
111, 8447–8462.
16 E. J. Parker, E. M. M. Bulloch, G. B. Jameson and C. Abell,
Biochemistry, 2001, 40, 14821–14828; C. M. Stephens and R. Bauerle,
J. Biol. Chem., 1991, 266, 20810–20817.
17 K. Duncan, S. Chaudhuri, M. S. Campbell and J. R. Coggins,
Biochem. J., 1986, 238, 475–483.
18 L. R. Schofield, B. F. Anderson, M. L. Patchett, G. E.
Norris, G. B. Jameson and E. J. Parker, Biochemistry, 2005, 44,
11950–11962.
19 E. D. Bergmann and I. Shahak, J. Chem. Soc., 1960, 462–463; J. A.
Stubbe and G. L. Kenyon, Biochemistry, 1972, 11, 338–345.
20 J. N. Baxter, A. S. Perlin and F. J. Simpson, Can. J. Biochem. Physiol.,
1959, 37, 199–209.
Steady state kinetics for P. furiosus DHQS with (3S)-3-
fluoroDAH7P. The standard assay reaction mixture contained
(3S)-3-fluoroDAH7P (10 to 680 mM) at pH 6.7 at 60 ◦C. The
mixture was preincubated at 60 ◦C for 5 min followed by addition
of 20 mL of P. furiosus DHQase (4.1 mg mL-1). After a further
minute of incubation, the reaction was initiated by the addition of
10 mL of P. furiosus DHQS (56 nM).
Steady state kinetics for E. coli DHQS with (3R)-3-
fluoroDAH7P. The standard assay reaction mixture contained
(3R)-3-fluoroDAH7P (2 to 160 mM), at pH 6.8 at 25 ◦C. The
mixture was preincubated at 25 ◦C for 1 min followed by addition
of 20 mL of E. coli DHQase (10 mg mL-1). After a further minute
21 N. Hasan and E. W. Nester, J. Biol. Chem., 1978, 253, 4999–5004.
22 P. A. Lanzetta, L. J. Alvarez, P. S. Reinach and O. A. Candia, Anal.
Biochem., 1979, 100, 95–97; H. H. Hess and J. E. Der, Anal. Biochem.,
1975, 63, 607–613.
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 2861–2867 | 2867
©