Su Dan, Yuan Hongling, Gadda Giovanni
Department of Chemistry, ‡Department of Biology, §Center for Diagnostics and Therapeutics, and ∥Center for Biotechnology and Drug Design, Georgia State University , Atlanta, Georgia 30302, United States.
Biochemistry. 2017 Dec 26;56(51):6677-6690. doi: 10.1021/acs.biochem.7b00958. Epub 2017 Dec 14.
Choline oxidase serves as a paradigm for alcohol oxidation catalyzed by flavin-dependent enzymes. In its active site, S101 is 4 Å from the flavin C4a atom on an extended loop. Enzyme variants substituted at S101 were generated in a previous study and investigated mechanistically [Yuan, H., and Gadda, G. (2011) Biochemistry 50, 770-779]. In this study, the typical ultraviolet-visible (UV-vis) absorption spectrum of oxidized flavin was observed for the S101C enzyme in HEPES, TES, or sodium phosphate, whereas an absorption spectrum suggesting the presence of a C4a-flavin adduct with cysteine was obtained in Tris-HCl at pH 8.0. pH titrations of the UV-vis absorption spectrum of the wild-type, S101A, S101C, and H99N enzymes in the presence and absence of Tris allowed for the determination of two pK values that define a pH range in which the C4a-S-cysteinyl flavin is stabilized. Inhibition studies and stopped-flow kinetics demonstrated that binding of protonated Tris in the active site of the S101C enzyme is required to form the C4a-S-cysteinyl flavin. Deuterium kinetic isotope effects and proton inventories of the S101C enzyme mixed in a stopped-flow spectrophotometer with Tris established a mechanism for the reversible formation of the C4a-S-cysteinyl flavin. This study provides a detailed mechanistic analysis of the reversible formation of a bicovalent C4a-S-cysteinyl-8α-N-histidyl flavin in choline oxidase, identifying an optimal pH range and a mechanistic rationale for the stabilization of de novo C4a-S-cysteinyl-flavins. Moreover, it presents an example of an intramolecular reaction of an enzyme-bound flavin without a substrate.
胆碱氧化酶是黄素依赖性酶催化醇氧化的一个范例。在其活性位点，S101位于延伸环上距黄素C4a原子4 Å处。在先前的一项研究中生成了在S101处被取代的酶变体，并进行了机制研究[袁，H.，和加达，G.（2011年）《生物化学》50，770 - 779]。在本研究中，在HEPES、TES或磷酸钠中观察到S101C酶具有氧化黄素的典型紫外可见（UV-vis）吸收光谱，而在pH 8.0的Tris - HCl中获得的吸收光谱表明存在与半胱氨酸形成的C4a - 黄素加合物。对野生型、S101A、S101C和H99N酶在有无Tris存在下的UV-vis吸收光谱进行pH滴定，从而确定了两个pK值，这两个pK值定义了一个C4a - S - 半胱氨酰黄素得以稳定的pH范围。抑制研究和停流动力学表明，S101C酶活性位点中质子化Tris的结合是形成C4a - S - 半胱氨酰黄素所必需的。在停流分光光度计中将S101C酶与Tris混合时的氘动力学同位素效应和质子丰度确定了C4a - S - 半胱氨酰黄素可逆形成的机制。本研究对胆碱氧化酶中双共价C4a - S - 半胱氨酰 - 8α - N - 组氨酰黄素的可逆形成进行了详细的机制分析，确定了一个最佳pH范围以及从头生成的C4a - S - 半胱氨酰黄素稳定化的机制原理。此外，它还展示了一个无底物时酶结合黄素的分子内反应实例。
