Mechanism and Catalytic Site Atlas

Introduction

Cysteine desulfurases are thought to share a common mechanism. The first step is the transimination through the substrate cysteine displacing Lys226 from Lys-PLP to form Cys-PLP via a tetrahedral geminal diamine intermediate. The Schiff base linkage and pyridine ring has a conjugated pi electron withdrawing effect, facilitating deprotonation of C-alpha. Deprotonation is most likely by a nearby histidine, His123. Kinetic, mutagenesis and inhibition studies have ruled out Lys226 or the cys substrate itself behaving as a general base. Cys364 acts as a general acid to protonate C4. The first committed step in the reaction is the now thiolate on Cys364 to attack as a nucleophile, forming the enzyme persulfide-covalent intermediate and ala-PLP intermediate. Subsequent proton transfers between His123 and C-alpha and C4 via ketimine and quinonoid intermediates occurs. Finally, the reverse transimination reaction takes place via aldimine and a tetrahedral geminal diamine intermediate restore the cofactor for catalysis. The reduced Cys364 is used in numerous sulphur containing reactions such as in biosynthesis of Fe-S clusters, biotin and thio-tRNA-nucleosides.

Out of the two proposed mechanisms, this is favoured due to clear evidence ruling out Lys226 and the cysteine substrate acting as general bases. That being said, more work is needed to be done to identify exactly the catalytic residues involved (His123 has been used here to act as both the general acid and base, may in fact be more residues with these roles). One of the substrate cysteines is apart of the enzyme, labelled Cys364 in both mechanism proposals.