Molecular basis for the stabilization and inhibition of 2, 3-dihydroxybiphenyl 1,2-dioxygenase by t-butanol.

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