Version 2 2020-09-02, 17:41Version 2 2020-09-02, 17:41
Version 1 2020-08-28, 12:05Version 1 2020-08-28, 12:05
journal contribution
posted on 2020-09-02, 17:41authored bySagar
M. Patel, Thomas G. Smith, Martha Morton, Kyle M. Stiers, Javier Seravalli, Stephen J. Mayclin, Thomas E. Edwards, John J. Tanner, Donald F. Becker
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 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.