AN ANALYSIS OF 3-MERCAPTOPYRUVATE SULFURTRANSFERASE (3-MST)
3-Mercaptopyruvate sulfurtransferase (MST, EC 126.96.36.199) is a ubiquitous enzyme. It is distributed in both prokaryotes and eukaryotes (Fig. 15.1), sequences for which can be found in the UniProt database (http://www.uniprot.org/) (The UniProt Consortium, 2015). Congenital MST deficiency causes mercaptolactate-cysteine disulfiduria (MCDU), which is registered at OMIM (OMIM entry %249650) (Amberger et al., 2015; Crawhall, 1978). This congenital disease was named after patients that excreted 3-mercaptolactate-cysteine disulfide (Fig. 15.2) (β-mercaptolactate-cysteine disulfide, S-(2-hydroxy-2-carboxyethylthio)cysteine) in their urine (Crawhall et al., 1968; Law and Fowler, 1976; Niederwieser et al., 1973). It is noteworthy that some cases were complicated by mental retardation (Crawhall et al., 1968; Law and Fowler, 1976). However, MCDU pathogenesis has not been clarified. In this chapter, we introduce the molecular properties, catalytic properties, physiological functions, and MST inhibitors that selectively inhibit persulfurated MST and propose the pharmacological usage of an enzyme inhibitor.
1.2 MST Structure and Catalytic Mechanism
Rat liver MST is made up of 295 amino acid residues, with a molecular weight of 32.8 kDa (Nagahara et al., 1999). MST and rhodanese have significant sequence similarity in which they share up to 66% identity, a conserved active site and a Cys catalytic residue (Spallarossa et al., 2004). The three-dimensional structure of MST is composed of two domains, a C-domain and an N-domain. The C-terminal domain contains the catalytic Cys247 residue that is located at the bottom of a shallow cavity with an orifice facing the interdomain space. The active site of MST undergoes a structural reshaping or “induced fit” to accommodate the substrate 3-MP, where rhodanese displays active site rigidity. The amino acid sequence of the active site loop of MST is different from rhodanese and does not form a semicircular conformation, but rather buries the catalytic Cys group in the protein core without an active site pocket on the protein surface (Spallarossa et al., 2004). Properties of the active site amino acids correlate with the ionic charge of the substrate 3-MP and function to electrostatically stabilize the substrate at the active site (Cipollone et al., 2007).
The formal catalytic mechanism of MST is not as well characterized as that of rhodanese. It appears to be a sequential kinetic process in which no sulfur-substituted enzyme intermediate is formed, distinct from rhodanese (Westley et al. 1983). Spallarossa et al. (2004) proposed the following catalytic cycle: the enzyme–substrate complex is formed, followed by isomerization of the Cys 247 covalent disulfide intermediate to a thiosulfoxide. This is followed by transfer of the sulfane sulfur to cyanide, producing a 3-cysteinyl-pyruvate adduct, which is then converted by nucleophilic reaction to free pyruvate and active enzyme. Alternatively, Nagahara et al. (1999) proposed the transfer of the 3-MP sulfur atom to the active site Cys to form a persulfide enzyme intermediate, followed by transfer of the sulfane sulfur to cyanide, thus forming SCN− and regenerating the active enzyme