Evidence for a stepwise mechanism of OMP decarboxylase [16]

Journal of the American Chemical Society

Volumn 121, Issue 29, 1999, Pages 6966-6967

  Ehrlich, Joel I ^a   Hwang, Chi Ching ^b   Cook, Paul F ^b   Blanchard, John S ^a  

^a ALBERT EINSTEIN COLLEGE OF MEDICINE   (United States)

^b UNIVERSITY OF OKLAHOMA   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

OROTIDINE 5' PHOSPHATE DECARBOXYLASE;

CATALYSIS; DECARBOXYLATION; ENZYME MECHANISM; ENZYME SUBSTRATE COMPLEX; ESCHERICHIA COLI; LETTER; NONHUMAN; REACTION ANALYSIS;

EID: 0033612737     PISSN: 00027863     EISSN: None     Source Type: Journal    
DOI: 10.1021/ja990737s     Document Type: Letter

References (22)

1. Radzicka, A.; Wolfenden, R. Science 1995, 267, 90-93.
2. Beak, P.; Siegel, B. J. Am. Chem. Soc. 1976, 98, 3601-3606.
3. Levine, H. L.; Brody, R. S.; Westheimer, F. H. Biochemistry 1980,19, 4994-4999.
4. Smiley, J. A.; Paneth, P.; O'Leary, M. H.; Bell, J. B.; Jones, M. E. Biochemistry 1991, 30, 6216-6223.
5. Lee, J. K.; Houk, K. N. Science 1997, 276, 942-945.
6. Smiley, J. A.; Jones, M. E. Biochemistry 1992, 31, 12162-12168.
7. Hermes, J. D.; Roeske, C. A.; O'Leary, M. M.; Cleland, W. W. Biochemistry 1982, 21, 5106-5114.
8. 600 = 0.6-0.8. Isopropyl-β-D-thiogalactoside was added to a final concentration of 100 μM, and cells were harvested after an additional 3 to 4 h. The cell pellet was resuspended (1:1 weight:volume) in 20 mM TEA pH 7.8, 1 mM DTT, Complete protease inhibitors (Boehringer-Mannheim), 1 mg/mL lysozyme, frozen at -20°C overnight and thawed at room temperature. The sample was disrupted by sonication and centrifuged. Streptomycin sulfate (pH 8) was added to the supernatant to a final concentration of 1% to precipitate nucleic acids. Following centrifugation, the supernatant (approximately 100 mL) was dialyzed 3-4 h against 12 L 20 mM TEA pH 7.8, 1 mM DTT, 50 mM NaCl, and ultracentrifuged (30K rpm). ODCase in the resulting supernatant was then purified to homogeneity by fast protein liquid chromatography (FPLC) on FastQ anion-exchange and phenyl-sepharose hydrophobic interaction columns (Pharmacia).
9. E. coli ODCase was inactivated (90%) by overnight dialysis against 0.5 mM EDTA. This is consistent with the recent suggestion (Miller, B. G.; Traut, T. W.; Wolfenden, R. J. Am. Chem. Soc. 1998, 120, 2666-2667) that the enzyme requires a metal cofactor.
10. 10b Reactions contained OMP, enzyme, 20 mM Hepes pL 7.5, and 0.5 mM DTT at room temperature.
11. (b) Brody, R. S.; Westheimer, F. H. J. Biol Chem. 1979, 254, 4238-4244.
12. 2 traps prior to analysis by isotope ratio mass spectrometry.
13. (b) O'Leary, M. H. Methods Enzymol. 1980, 64, 83-104.
14. 2-7
15. 4 represent "net" rate constants that describe the entire solvent-sensitive portion of the reaction coordinate, which may formally consist of a single or multiple microscopic steps.
16. 2).
17. 3} array satisfied eqs 1, 2, and 3, and yielded values for the forward commitments for decarboxylation smaller than unity, as expected.
18. (b) Quinn, D. M.; Sutton, L. D. In Enzyme Mechanism from Isotope Effects; Cook, P. F., Ed.; CRC Press: Boca Raton, Florida, 1991;73-126.
19. (c) Karsten, W. E.; Gavva, S. R.; Park S. H.; Cook P. F. Biochemistry 1995, 34, 3253-60.
20. (d) Grissom, C. B.; Cleland, W. W. Biochemistry 1988, 27, 2927-34.
21. 2 with protonation occurring at the C2 carbonyl is also consistent with these results. We elect to interpret the data in the context of the Houk mechanism on the basis of its more favorable predicted thermodynamics.
22. Tao, W.; Grubmeyer, C.; Blanchard, J. S. Biochemistry 1996, 35, 14-21.