Thesis Abstract

Abstract
3-Mercaptopyruvate sulfurtransferase (3-MST) is an enzyme that plays a crucial role in the maintenance of redox homeostasis and hydrogen sulfide (H2S) metabolism in various tissues and organs. This enzyme is primarily responsible for catalyzing the transfer of a sulfur atom from 3-mercaptopyruvate to various acceptors, leading to the formation of persulfides and polysulfides. Through this process, 3-MST contributes to the regulation of H2S levels in the body, which is essential for a wide range of physiological functions. Studies have shown that 3-MST is expressed in various tissues, with particularly high levels in the brain, liver, and kidney. Its expression and activity have been implicated in the regulation of cellular redox status, protection against oxidative stress, and modulation of signaling pathways involved in inflammation and apoptosis. Dysregulation of 3-MST activity has been linked to several pathological conditions, including cardiovascular diseases, neurodegenerative disorders, and cancer. Recent research has shed light on the structural and functional properties of 3-MST, revealing key insights into its catalytic mechanism and regulatory processes. The enzyme's active site contains a conserved cysteine residue that is essential for its sulfur transferase activity, highlighting the importance of redox regulation in modulating 3-MST function. Additionally, post-translational modifications, such as phosphorylation and S-sulfhydration, have been shown to influence 3-MST activity and localization within the cell. Pharmacological modulation of 3-MST activity has emerged as a potential therapeutic strategy for various diseases. Small molecule inhibitors and activators targeting 3-MST have been developed to manipulate H2S levels and redox balance in a tissue-specific manner. These compounds hold promise for the treatment of conditions associated with H2S dysregulation, such as hypertension, diabetes, and neurodegeneration. In conclusion, 3-Mercaptopyruvate sulfurtransferase is a key enzyme involved in H2S metabolism and redox regulation in the body. Understanding the biochemical and physiological functions of 3-MST is essential for elucidating its role in health and disease. Further research into the mechanisms of 3-MST regulation and the development of targeted therapies may pave the way for novel treatment strategies for a variety of disorders.

Thesis Overview

INTRODUCTION AND LITERATURE REVIEW

INTRODUCTION

One of the major metabolic enzymes that have gained so much interest of scientists is 3-Mercaptopyruvate sulfurtransferase (3-MST). This enzyme occurs widely in nature (Bordo, 2002 and Jarabak, 1981). It has been reported in several organisms ranging from humans to rats, fishes and insects. It is a mitochondrial enzyme which has been concerned in the detoxification of cyanide, a potent toxin of the mitochondrial respiratory chain (Nelson et al., 2000). Among the several metabolic enzymes that carry out xenobiotic detoxification, 3-mercaptopyruvate sulfurtransferase is of utmost importance. 3-mercaptopyruvate sulfurtransferase functions in the detoxifications of cyanide; mediation of sulfur ion transfer to cyanide or to other thiol compounds.(Vandenet al., 1967).It is also required for the biosynthesis of thiosulfate. In combination with cysteine aminotransferase, it contributes to the catabolism of cysteine and it is important in generating hydrogen sulphide in the brain, retina and vascular endothelial cells (Shibuyaet al., 2009). It also acquired different functions such as a redox regulation (maintenance of cellular redox homeostasis) and defense against oxidative stress, in the atmosphere under oxidizing conditionsNagaharaet al (2005).

Hydrogen sulphide (H2S) is an important synaptic modulator, signalling molecule, smooth muscle contractor and neuroprotectant (Hosokiet al., 1997). Its production by the 3-mercaptopyruvate sulfurtransferase and cysteine aminotransferase pathways is regulated by calcium ions (Hosokiet al., 1997). Organisms that are exposed to cyanide poisoning usually have this enzyme in them. This could be in food as in the cyanogenicglucosides being consumed. It has been studied from variety of sources, which include bacteria, yeasts, plants, and animals (Marcus Wischik, 1998).

Cyanide could be released into the bark of trees as a defence mechanism. There are array of defensive compounds that make their parts (leaves, flowers, stems, roots and fruits) distasteful or poisonous to predators. In response, however, the animals that feed on them have evolved over successive generations a range of measures to overcome these compounds and can eat the plant safely. The tree trunk offers a clear example of the variety of defences available to plants (Marcus Wischik, 1998). Oryctes rhinoceros larva is one of the organisms that are also exposed to cyanide toxicity because of the environment they are found.

1.2. 3-MERCAPTOPYRUVATE SULFURTRANSFERASE

3-Mercaptopyruvate sulfurtransferase (EC. 2.8.1.2), is a member of the group, Sulfurtransferases (EC 2.8.1.1 – 5), which are widely distributed enzymes of prokaryotes and eukaryotes (Bordoand Bork, 2002). 3-Mercaptopyruvate Sulfurtransferase is an enzyme that is part of the cysteine catabolic pathway. The enzyme catalyzes the conversion 3-mercaptopyruvate to pyruvate and H2S (Shibuya et al., 2009). The deficiency of this enzyme will result in elevated urine concentrations of 3-mercaptopyruvate as well as of 3-mercaptolactate, both in the form of disulfides with cysteine(Crawhallet al., 1969). It catalyzes the chemical reaction:

3-mercaptopyruvate + cyanide à pyruvate + thiocyanate

3-mercaptopyruvate + thiolà pyruvate + hydrogen sulphide (Sorbo 1957).

It transfers sulfur-containing groups and participates in cysteine metabolism (Shibuya et al., 2013). This enzyme catalyzes the transfer of sulfane sulphur from a donor molecule, such as thiosulfate or 3- mercaptopyruvate, to a nucleophile acceptor, such as cyanide or mercptoethanol.3-mercaptopyruvate is the known sulphur-donor substrate for 3-mercaptopyruvate sulfurtransferase (Porter & Baskin, 1995). 3-mercaptopyruvate sulfurtransferase is believed to function in the endogenous cyanide (CN) detoxification system because it is capable of transferring sulphur from 3-mercaptopyruvate (3-MP) to cyanide (CN), forming the less toxic thiocyanate (SCN) (Hylin and Wood, 1959). It is an important enzyme for the synthesis of hydrogen sulphide (H2S) in the brain (Shibuya et al., 2009).

The systematic name of this enzyme class is 3-mercaptopyruvate: cyanide sulfurtransferase. It is also called beta-mercaptopyruvatesulfurtransferase(Vachek and Wood, 1972).It is one of three known H2S producing enzymes in the body (Hylin and Wood, 1959). It is primarily localised in the mitochondria (Cipolloneet al., 2008). The expression levels of 3-MST in the brain during the fetal and postnatal periods are higher than those in the adult brain (unpublished data) although the promoter region shows characteristics of a typical housekeeping gene (Nagaharaet al., 2004). The observation is supported by the finding that3-MST expression in the cerebellum is decreased during the adult period (Shibuya et al., 2013). On the other hand, its expression level in the lung decreases from the perinatal period. These facts suggest that 3-MST could function in the fetal and postnatal brain. It was reported that serotonin signaling via the 5-HT1A receptor in the brain during the early developmental stage plays a critical role in the establishment of innate anxiety during the early developmental stage (Richardson-Jones et al., 2011).

In rat, 3-MST possesses 2 redox-sensing molecular switches (Nagahara and Katayama, 2005). A catalytic-site cysteine and an intersubunitdisulfide bond serve as a thioredoxin-specific molecular switch (Nagaharaet al., 2007). The intermolecular switch is not observed in prokaryotes and plants, which emerged into the atmosphere under reducing conditions (Nagahara, 2013). As a result, it acquired different functions such as a redox regulation (maintenance of cellular redox homeostasis) and defense against oxidative stress, in the atmosphere under oxidizing conditions (Nagaharaet al., 2005).

Moreover, 3-MST can produce H2S (or HS−) as a biofactor (Shibuya et al., 2009), which cystathionine β-synthase and cystathionine γ-lyase also can generate (Abe and Kimura, 1996). Interestingly 3-MST can uniquely produce SOx in the redox cycle of persulfide formed at the low-redox catalytic-site cysteine (Nagaharaet al., 2012). As an alternate hypothesis on the pathogenesis of the symptoms, H2S (or HS−) and/or SOxcould suppress anxiety-like behavior, and therefore, defects in these molecules could increase anxiety-like behavior. However, no microanalysis method has been established to quantify H2S (or HS−) and SOxat the physiological level (Ampolaet al., 1969).MCDU was first recognized and reported in 1968 as an inherited metabolic disorder caused by congenital 3-MST insufficiency or deficiency. Most cases were associated with mental retardation (Ampolaet al, 1969) while the pathogenesis remains unknown.

Human MCDU was reported to be associated with behavioral abnormalities, mental retardation (Crawhall, 1985), hypokinetic behaviour, and grand mal seizures and anomalies (flattened nasal bridge and excessively arched palate) (Ampolaet al, 1969); however, the pathogenesis has not been clarified since MCDU was recognized more than 40 years ago. Macroscopic anomalies were associated in 1 case (Ampolaet al, 1969); however, this could be an accidental combination. 3-MST deficiency also induced higher brain dysfunction in mice without macroscopic and microscopic abnormalities in the brain. 3-MST seems to play a critical role in the central nervous system, i.e., to establish normal anxiety (Richardson et al., 2011)

1.2.1. DISTRIBUTION OF 3-MST

3-MST is widely distributed in prokaryotes and eukaryotes (Jarabak, 1981). It is localized in the cytoplasm and mitochondria, but not all cells contain 3-MST (Nagaharaet al., 1998).

1.2.2. OCCURRENCE

Human mercaptopyruvatesulfurtransferase (MPST; EC. 2.8.1.2) belongs to the family of sulfurtransferases (Vandenet al., 1967). These enzymes catalyze the transfer of sulfur to a thiophilic acceptor (Sorbo 1957), where MPST has a preference for 3-mercapto sulfurtransferase as the sulfur-donor. MPST plays a central role in both cysteine degradation and cyanide detoxification. In addition, deficiency in MPST activity has been proposed to be responsible for a rare inheritable disease known as mercaptolactate-cysteine disulfiduria (MCDU) (Hannestadet al, 2006).

1.2.3. MECHANISMS OF ACTION

3-Mercaptopyruvate sulfurtransferasecatalyzes the reaction from mercaptopyruvate (SHCH2C (= O)COOH)) to pyruvate (CH3C(= O)COOH) in cysteine catabolism (Vackek and Wood, 1972). The enzyme is widely distributed in prokaryotes and eukaryotes (Jarabak, 1981). This disulfide bond serves as a thioredoxin-specific molecular switch. On the other hand, a catalytic-site cysteine is easily oxidized to form a low-redox potential sulfenate which results in loss of activity (Nahagaraet al., 2005). Then, thioredoxin can uniquely restore the activity (Nagahara, 2013). Thus, a catalytic site cysteine contributes to redox-dependent regulation of 3-MST activity serving as a redox-sensing molecular switch (Nahagara, 2013). These findings suggest that 3-MST serves as an antioxidant protein and partly maintain cellular redox homeostasis. Further, it was proposed that 3-MST can produce hydrogen sulphide (H2S) by using a persulfurated acceptor substrate (Shibuya et al, 2009). As an alternative functional diversity of 3-MST, it has been recently demonstrated in-vitro that 3-MST can produce sulfur oxides (SOx) in the redox cycle of persulfide (S-S-) formed at the catalytic site of the reaction intermediate (Nagaharaet al, 2012).

1.2.4. MOLECULAR FORMULA AND MOLECULAR WEIGHT

The molecular formula of 3-MST is C3H4O3S (Vachek and Wood, 1972).

3-MST has a molecular weight of 120.127g/mol or 23800 Daltons (as summarized by PubChem compound).