Molecular basis for the stabilization and inhibition of 2, 3-dihydroxybiphenyl 1,2-dioxygenase by t-butanol.
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 Km for dioxygen was 1280 +/- 70 microM, which is at least 2 orders of magnitude higher than that reported for catechol 2,3-dioxygenases. Km and Kd for DHB were 22 +/- 2 and 8 +/- 1 microM, 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.
Vaillancourt
F H
FH
Department of Biochemistry, Pavillon Marchand, Université Laval, Québec City, P.Q. G1K 7P4, Canada.
Han
S
S
Fortin
P D
PD
Bolin
J T
JT
Eltis
L D
LD
D001673:Biodegradation, Environmental; D001713:Biphenyl Compounds / Q000378:metabolism; D016956:Burkholderia cepacia / Q000201:enzymology; D002396:Catechols / Q000378:metabolism; D049308:Dioxygenases; D004795:Enzyme Stability / Q000187:drug effects; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D010105:Oxygenases / Q000037:antagonists & inhibitors / Q000187:drug effects*; D014961:X-Ray Diffraction; D020002:tert-Butyl Alcohol / Q000494:pharmacology*
D001713:Biphenyl Compounds; D002396:Catechols; D010105:Oxygenases; D049308:Dioxygenases; C074272:2,3-dihydroxybiphenyl oxygenase; D020002:tert-Butyl Alcohol