Cautionary Tale of Using Tris(alkyl)phosphine Reducing Agents with NAD⁺-Dependent Enzymes

Abstract

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 Δ 1 -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 ( 1 H, 13 C, and 31 P) 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 Å 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.

Martin’s Phosphino-Triol Revisited: Unexpected P–C Bond Cleavage Reactions and Their Suppression via Complexation of Al3+ and Sc3+

“…Tris­(hydroxymethyl)­phosphine, P­(CH 2 OH) 3 , is a well-known and easily prepared C 3 -symmetric phosphino-triol, which is routinely employed in industry and academia, not only for the preparation of water-soluble metal complexes but also as a flame retardant, a multipurpose reductant, and a medicinal O 2 -scavanger . The chemistry of the higher homologues P­[(CH 2 ) n OH] 3 ( n = 2, 3) has been explored as well, albeit only tris­(hydroxypropyl)­phosphine ( n = 3) is regularly used as a mild reductant for the reductive cleavage of disulfide bonds . In comparison to these alkyl phosphino-triols, our knowledge on related hydroxylated aryl phosphines is still limited; i.e., only a few reports on the two most simple Ph 3 P-based phosphino-triols, namely, P­(C 6 H 4 - o -OH) 3 and P­(C 6 H 4 - o -CH 2 OH) 3 (and their derivatives), have been published to date (see Scheme ).…”

Active site remodeling in tumor-relevant IDH1 mutants drives distinct kinetic features and potential resistance mechanisms

“…Support for a puckered ring during the transition from oxidized to reduced cofactor has been described in a structure of horse liver alcohol dehydrogenase, where the puckered nicotinamide ring was in a quasi-boat conformation with bond angles and distances suggesting a strained molecule that could mimic a transition state (Plapp & Ramaswamy, 2012). Interestingly, NAD(P)-adducts with reducing agents have been reported previously, including with TCEP (Patel et al, 2020) and also with DTT (Paidimuddala et al, 2018), and were often found to have a more puckered nicotinamide ring. S2), suggestive of a transition-state-like conformation.…”

Structure of the LarB–Substrate Complex and Identification of a Reaction Intermediate during Nickel-Pincer Nucleotide Cofactor Biosynthesis

“…Reduction of NAD + to yield NADH generates the well-known absorbance at 340 nm and an analogous absorbance occurs after hydride addition to NaAD (Figure S3A,B). It is also established that thiols and a variety of nucleophiles including tris­(alkyl)­phosphine reductants can reversibly add to C4 of the pyridinium ring of nicotinamides to generate similar absorbances. − Thus, we reasoned that a Cys221-pyridine adduct (intermediates 1 and 2, Figure ) would also be observed by UV–vis spectroscopy if formed at sufficient abundance. Indeed, we demonstrated that an absorbance with a maximum near 370 nm is generated when NAD + was titrated into the WT or S127A forms of LarB, with all solutions argon purged to prevent carboxylation (Figure S3C,D).…”