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J Biol Chem, Vol. 273, Issue 52, 34887-34895, December 25, 1998

Molecular Basis for the Stabilization and Inhibition of 2,3-Dihydroxybiphenyl 1,2-Dioxygenase by t-Butanol

Frédéric H. Vaillancourt, Seungil Han^¶, Pascal D. Fortin, Jeffrey T. Bolin^¶, and Lindsay D. Eltis

From the  Department of Biochemistry, Pavillon Marchand, Université Laval, Québec City, P.Q. G1K 7P4, Canada and the ^¶ Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907-1392

The steady-state cleavage of catechols by 2,3-dihydroxybiphenyl 1,2-dioxygenase (DHBD), the extradiol dioxygenase of the biphenyl biodegradation pathway, was investigated using a highly active, anaerobically purified preparation of enzyme. The kinetic data obtained using 2,3-dihydroxybiphenyl (DHB) fit a compulsory order ternary complex mechanism in which substrate inhibition occurs. The K_m for dioxygen was 1280 ± 70 µM, which is at least 2 orders of magnitude higher than that reported for catechol 2,3-dioxygenases. K_m and K_d for DHB were 22 ± 2 and 8 ± 1 µM, respectively. DHBD was subject to reversible substrate inhibition and mechanism-based inactivation. In air-saturated buffer, the partition ratios of catecholic substrates substituted at C-3 were inversely related to their apparent specificity constants. Small organic molecules that stabilized DHBD most effectively also inhibited the cleavage reaction most strongly. The steady-state kinetic data and crystallographic results suggest that the stabilization and inhibition are due to specific interactions between the organic molecule and the active site of the enzyme. t-Butanol stabilized the enzyme and inhibited the cleavage of DHB in a mixed fashion, consistent with the distinct binding sites occupied by t-butanol in the crystal structures of the substrate-free form of the enzyme and the enzyme-DHB complex. In contrast, crystal structures of complexes with catechol and 3-methylcatechol revealed relationships between the binding of these smaller substrates and t-butanol that are consistent with the observed competitive inhibition.

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