Vaillancourt F H, Han S, Fortin P D, Bolin J T, Eltis L D
Department of Biochemistry, Pavillon Marchand, Université Laval, Québec City, P.Q. G1K 7P4, Canada.
J Biol Chem. 1998 Dec 25;273(52):34887-95. doi: 10.1074/jbc.273.52.34887.
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.
利用高活性、经厌氧纯化的联苯双加氧酶制剂，研究了联苯生物降解途径中的间位二醇双加氧酶——2,3 -二羟基联苯1,2 -双加氧酶（DHBD）对儿茶酚的稳态裂解作用。使用2,3 -二羟基联苯（DHB）获得的动力学数据符合存在底物抑制的强制顺序三元复合物机制。氧气的Km值为1280±70微摩尔，这比儿茶酚2,3 -双加氧酶报道的值至少高2个数量级。DHB的Km和Kd分别为22±2和8±1微摩尔。DHBD会受到可逆的底物抑制和基于机制的失活作用。在空气饱和缓冲液中，C - 3位被取代的儿茶酚底物的分配比与其表观特异性常数呈负相关。最有效地稳定DHBD的小分子对裂解反应的抑制作用也最强。稳态动力学数据和晶体学结果表明，这种稳定作用和抑制作用是由于有机分子与酶活性位点之间的特异性相互作用。叔丁醇以混合方式稳定酶并抑制DHB的裂解，这与叔丁醇在无底物形式的酶和酶 - DHB复合物的晶体结构中占据不同的结合位点一致相。相比之下，儿茶酚和3 -甲基儿茶酚复合物的晶体结构揭示了这些较小底物与叔丁醇的结合之间的关系，这与观察到的竞争性抑制作用一致。
