Chen Lin, Lyubimov Artem Y, Brammer Leighanne, Vrielink Alice, Sampson Nicole S
Department of Chemistry, Stony Brook University, Stony Brook, New York 11794, USA.
Biochemistry. 2008 May 13;47(19):5368-77. doi: 10.1021/bi800228w. Epub 2008 Apr 15.
The usage by enzymes of specific binding pathways for gaseous substrates or products is debated. The crystal structure of the redox enzyme cholesterol oxidase, determined at sub-angstrom resolution, revealed a hydrophobic tunnel that may serve as a binding pathway for oxygen and hydrogen peroxide. This tunnel is formed by a cascade of conformational rearrangements and connects the active site with the exterior surface of the protein. To elucidate the relationship between this tunnel and gas binding and release, three mutant enzymes were constructed to block the tunnel or its putative gate. Mutation of the proposed gating residue Asn485 to Asp or tunnel residue Phe359 or Gly347 to Trp or Asn reduces the catalytic efficiency of oxidation. The K mO 2 increases from 300 +/- 35 microM for the wild-type enzyme to 617 +/- 15 microM for the F359W mutant. The k cat for the F359W mutant-catalyzed reaction decreases 13-fold relative to that of the wild-type-catalyzed reaction. The N485D and G347N mutants could not be saturated with oxygen. Transfer of hydride from the sterol to the flavin prosthetic group is no longer rate-limiting for these tunnel mutants. The steady-state kinetics of both wild-type and tunnel mutant enzymes are consistent with formation of a ternary complex of steroid and oxygen during catalysis. Furthermore, kinetic cooperativity with respect to molecular oxygen is observed with the tunnel mutants, but not with the wild-type enzyme. A rate-limiting conformational change for binding and release of oxygen and hydrogen peroxide, respectively, is consistent with the cooperative kinetics. In the atomic-resolution structure of F359W, the indole ring of the tryptophan completely fills the tunnel and is observed in only a single conformation. The size of the indole is proposed to limit conformational rearrangement of residue 359 that leads to tunnel opening in the wild-type enzyme. Overall, these results substantiate the functional importance of the tunnel for substrate binding and product release.
酶对气态底物或产物使用特定结合途径这一现象存在争议。以亚埃分辨率测定的氧化还原酶胆固醇氧化酶的晶体结构揭示了一条疏水通道，该通道可能作为氧气和过氧化氢的结合途径。这条通道由一系列构象重排形成，将活性位点与蛋白质的外表面相连。为阐明这条通道与气体结合和释放之间的关系，构建了三种突变酶来阻断通道或其假定的门控位点。将假定的门控残基Asn485突变为Asp，或将通道残基Phe359或Gly347突变为Trp或Asn，会降低氧化的催化效率。野生型酶的KmO₂为300±35 μM，而F359W突变体的KmO₂增加到617±15 μM。F359W突变体催化反应的kcat相对于野生型催化反应降低了13倍。N485D和G347N突变体无法被氧气饱和。对于这些通道突变体，从甾醇向黄素辅基的氢化物转移不再是限速步骤。野生型和通道突变体酶的稳态动力学均与催化过程中类固醇和氧气形成三元复合物一致。此外，通道突变体观察到了对分子氧的动力学协同性，而野生型酶则未观察到。分别对于氧气和过氧化氢的结合与释放存在限速构象变化，这与协同动力学一致。在F359W的原子分辨率结构中，色氨酸的吲哚环完全填充了通道，且仅以单一构象存在。吲哚的大小被认为限制了359位残基的构象重排，而这种重排在野生型酶中会导致通道打开。总体而言，这些结果证实了通道对于底物结合和产物释放的功能重要性。
