Basran Jaswir, Fullerton Stephen, Leys David, Scrutton Nigel S
Department of Biochemistry, University of Leicester, UK.
Biochemistry. 2006 Sep 19;45(37):11151-61. doi: 10.1021/bi061094d.
Residues His-225 and Tyr-259 are located close to the FAD in the dehydrogenase active site of the bifunctional dimethylglycine oxidase (DMGO) of Arthrobacter globiformis. We have suggested [Leys, D., Basran, J., and Scrutton, N. S. (2003) EMBO J. 22, 4038-4048] that these residues are involved in abstraction of a proton from the substrate amine group of dimethylglycine prior to C-H bond breakage and FAD reduction. To investigate this proposal, we have isolated two mutant forms of DMGO in which (i) His-225 is replaced with Gln-225 (H225Q mutant) and (ii) Tyr-259 is replaced with Phe-259 (Y259F mutant). Both mutant enzymes retain the ability to oxidize substrate, but the steady-state turnover of the Y259F mutant is attenuated more than 200-fold. Only modest changes in kinetic parameters are observed for the H225Q mutant during steady-state turnover. Stopped-flow studies indicate that the rate of FAD reduction in the Y259F enzyme is substantially impaired by a factor of approximately 1500 compared with that of the wild-type enzyme, suggesting a key role for this residue in the reductive half-reaction of the enzyme. The kinetics of FAD reduction in the H225Q enzyme are complex and involve three discrete kinetic phases that are attributed to different conformational states of this mutant, evidence for which is provided by crystallographic analysis. Neither the H225Q enzyme nor the Y259F enzyme stabilizes the FADH(2)-iminium charge-transfer complex observed previously in stopped-flow studies with the wild-type enzyme. Our studies are consistent with a key role for Tyr-259, but not His-225, in deprotonation of the substrate amine group prior to FAD reduction. We infer that residue His-225 is likely to modulate the acid-base properties of Tyr-259 by perturbing the pK(a) of Tyr-259 and thus fine-tunes the reaction chemistry to facilitate proton abstraction under physiological conditions. Our data are discussed in the context of the crystallographic data for DMGO and also in relation to contemporary mechanisms for flavoprotein-catalyzed oxidation of amine substrates.
在球形节杆菌双功能二甲基甘氨酸氧化酶（DMGO）的脱氢酶活性位点中，组氨酸-225（His-225）和酪氨酸-259（Tyr-259）残基靠近黄素腺嘌呤二核苷酸（FAD）。我们曾提出[Leys, D., Basran, J., and Scrutton, N. S. (2003) EMBO J. 22, 4038 - 4048]，这些残基在碳氢键断裂和FAD还原之前，参与从二甲基甘氨酸的底物胺基上夺取一个质子的过程。为了研究这一推测，我们分离出了两种DMGO突变体形式，其中（i）His-225被谷氨酰胺-225（Gln-225）取代（H225Q突变体），（ii）Tyr-259被苯丙氨酸-259（Phe-259）取代（Y259F突变体）。两种突变酶都保留了氧化底物的能力，但Y259F突变体的稳态周转数降低了200多倍。在稳态周转过程中，H225Q突变体的动力学参数仅发生了适度变化。停流研究表明，与野生型酶相比，Y259F酶中FAD还原速率大幅受损，约为野生型酶的1/1500，这表明该残基在酶的还原半反应中起关键作用。H225Q酶中FAD还原的动力学很复杂，涉及三个不同的动力学阶段，这归因于该突变体的不同构象状态，晶体学分析为这一点提供了证据。H225Q酶和Y259F酶都不能稳定先前在野生型酶的停流研究中观察到的FADH(2)-亚胺离子电荷转移复合物。我们的研究结果表明，在FAD还原之前，Tyr-259而非His-225在底物胺基的去质子化过程中起关键作用。我们推断，His-225残基可能通过扰动Tyr-259的pK(a)来调节其酸碱性质，从而在生理条件下微调反应化学过程以促进质子夺取。我们的数据将结合DMGO的晶体学数据以及黄素蛋白催化胺底物氧化的当代机制进行讨论。
