Cautionary Tale of Using Tris(alkyl)phosphine Reducing Agents with NAD+-Dependent Enzymes
| العنوان: | Cautionary Tale of Using Tris(alkyl)phosphine Reducing Agents with NAD+-Dependent Enzymes |
|---|---|
| المؤلفون: | Kyle M. Stiers, Thomas E. Edwards, Martha D. Morton, Javier Seravalli, John J. Tanner, Stephen J. Mayclin, Donald F. Becker, Thomas G. Smith, Sagar M. Patel |
| المصدر: | Biochemistry. 59:3285-3289 |
| بيانات النشر: | American Chemical Society (ACS), 2020. |
| سنة النشر: | 2020 |
| مصطلحات موضوعية: | Tris, chemistry.chemical_compound, Cofactor binding, chemistry, Reducing agent, TCEP, Dehydrogenase, NAD+ kinase, Biochemistry, Medicinal chemistry, Dithiothreitol, Phosphine |
| الوصف: | Protein biochemistry protocols typically include disulfide bond reducing agents to guard against unwanted thiol oxidation and protein aggregation. Commonly used disulfide bond reducing agents include dithiothreitol, β-mercaptoethanol, glutathione, and the tris(alkyl)phosphine compounds tris(2-carboxyethyl)phosphine (TCEP) and tris(3-hydroxypropyl)phosphine (THPP). While studying the catalytic activity of the NAD(P)H-dependent enzyme Δ1-pyrroline-5-carboxylate reductase, we unexpectedly observed a rapid non-enzymatic chemical reaction between NAD+ and the reducing agents TCEP and THPP. The product of the reaction exhibits a maximum ultraviolet absorbance peak at 334 nm and forms with an apparent association rate constant of 231-491 M-1 s-1. The reaction is reversible, and nuclear magnetic resonance characterization (1H, 13C, and 31P) of the product revealed a covalent adduct between the phosphorus of the tris(alkyl)phosphine reducing agent and the C4 atom of the nicotinamide ring of NAD+. We also report a 1.45 A resolution crystal structure of short-chain dehydrogenase/reductase with the NADP+-TCEP reaction product bound in the cofactor binding site, which shows that the adduct can potentially inhibit enzymes. These findings serve to caution researchers when using TCEP or THPP in experimental protocols with NAD(P)+. Because NAD(P)+-dependent oxidoreductases are widespread in nature, our results may be broadly relevant. |
| تدمد: | 1520-4995 0006-2960 |
| URL الوصول: | https://explore.openaire.eu/search/publication?articleId=doi_________::a557a70f53b831298427794fff6c97c7 https://doi.org/10.1021/acs.biochem.0c00490 |
| حقوق: | OPEN |
| رقم الأكسشن: | edsair.doi...........a557a70f53b831298427794fff6c97c7 |
| قاعدة البيانات: | OpenAIRE |
Find this issue from the American Chemical Society | تدمد: | 15204995 00062960 |
|---|