Project Abstract

Abstract
3-Mercaptopyruvate sulfurtransferase (3-MST) is a crucial enzyme involved in the regulation of hydrogen sulfide (H2S) levels in the body. This enzyme catalyzes the transfer of a sulfur atom from 3-mercaptopyruvate to various acceptor molecules, leading to the production of H2S. The physiological functions of 3-MST and its product, H2S, have gained significant attention due to their roles in various biological processes, including cardiovascular regulation, inflammation, and cellular signaling. This research project aims to provide a comprehensive analysis of 3-MST, focusing on its structure, function, regulation, and physiological significance. The structural features of 3-MST, including its active site residues and catalytic mechanism, will be explored using bioinformatics tools and molecular modeling techniques. Additionally, the interactions of 3-MST with its substrates and inhibitors will be investigated to elucidate the molecular basis of its enzymatic activity. Furthermore, the regulatory mechanisms of 3-MST expression and activity will be examined to understand how this enzyme is controlled in different physiological conditions. The impact of post-translational modifications, cofactors, and interacting proteins on 3-MST function will be evaluated to uncover the complex regulatory networks governing H2S metabolism in the body. Moreover, the physiological significance of 3-MST will be discussed in the context of its role in H2S production and signaling. The implications of 3-MST-mediated H2S production in cardiovascular health, inflammation, and neuroprotection will be highlighted, emphasizing the therapeutic potential of targeting 3-MST for the treatment of various diseases. Overall, this research project will contribute to a better understanding of the biochemical and physiological functions of 3-MST, shedding light on its significance in health and disease. By elucidating the molecular mechanisms underlying 3-MST activity and regulation, this study may pave the way for the development of novel therapeutic strategies targeting the H2S signaling pathway for the treatment of cardiovascular disorders, inflammatory conditions, and other diseases associated with dysregulated sulfur metabolism.

Project Overview

1.0. INTRODUCTION AND LITERATURE REVIEW

1.1. 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).