Biochemical Discrimination between Selenium and Sulfur 2: Mechanistic Investigation of the Selenium Specificity of Human Selenocysteine Lyase

Abstract

Selenium is an essential trace element incorporated into selenoproteins as selenocysteine. Selenocysteine (Sec) lyases (SCLs) and cysteine (Cys) desulfurases (CDs) catalyze the removal of selenium or sulfur from Sec or Cys, respectively, and generally accept both substrates. Intriguingly, human SCL (hSCL) is specific for Sec even though the only difference between Sec and Cys is a single chalcogen atom.The crystal structure of hSCL was recently determined and gain-of-function protein variants that also could accept Cys as substrate were identified. To obtain mechanistic insight into the chemical basis for its substrate discrimination, we here report time-resolved spectroscopic studies comparing the reactions of the Sec-specific wild-type hSCL and the gain-of-function D146K/H389T variant, when given Cys as a substrate. The data are interpreted in light of other studies of SCL/CD enzymes and offer mechanistic insight into the function of the wild-type enzyme. Based on these results and previously available data we propose a reaction mechanism whereby the Sec over Cys specificity is achieved using a combination of chemical and physico-mechanical control mechanisms.

Exploring the selenium-over-sulfur substrate specificity and kinetics of a bacterial selenocysteine lyase

“…For some enzymes, large changes in selenium recognition are due to a single point mutation. As stated previously, a D146K mutation of hSCL resulted in CDS activity (50,51). Likewise, a H240N variant of Astragalus bisulcatus CysRS was known to discriminate against selenocysteine (56).…”

“…It is known that the selenocysteine specificity of hSCL is strongly governed by D146 (50,51). With this in mind, we wondered if this "single-residue" property held true in SclA.…”

“…An investigation into the selenium-oversulfur specificity mechanism of human SCL (hSCL) has shed some light on this topic. A sitedirected mutagenesis approach found that mutation of the aspartic acid at amino acid position 146 (D146) to a lysine (D146K) resulting in novel activity towards L-cysteine (50,51). This implied that D146 is either the sole determinant of selenium specificity or at least plays a significant role in the mechanism.…”

“…This implied that D146 is either the sole determinant of selenium specificity or at least plays a significant role in the mechanism. Specifically, the authors proposed that, while a protonated thiol could not react with the thiol of L-cysteine, the nucleophilic selenolate anion of L-selenocysteine may deprotonate the catalytic cysteine residue's thiol which could then attack the electrophilic selenol group, causing the reaction to proceed with high specificity for selenium (51). In this case, it is believed that the nearby carboxylate anion of D146 serves to increase the pKa of C388 (the catalytic cysteine) through a like-charge destabilization of its thiolate anion; thus, the protonated thiol species would dominate, allowing only reaction with Lselenocysteine.…”

Biological Chemistry of Hydrogen Selenide

“…Sulfur is approximately 10 5 times more abundant in the human body than selenium, but the latter element is selected for certain biological functions [27]. It is particularly remarkable that this is achieved, as there are no other two main-group elements that exhibit more similar physical and chemical properties [28].…”