In enzymology,a 3-mercaptopyruvate sulfurtransferase (EC 188.8.131.52) is an enzyme that catalyses the chemical reactions of 3-mercaptopyruvate.This enzyme belong to the family of transferases, specifically the sulfurtransferase [Yadav PK,2013].This enzyme participates in cysteine metabolism. It is encoded by the MPST gene. The enzyme is of interest because it provides a pathway for detoxification of cyanide, especially since it occurs widely in the cytosol and distributed broadly [Patterson SE,2016]. 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 utmost importance
In addition,3mst is localized to neurons, and the level of bound sulfane sulphur, the precursor of H2S, are greatly increased in the cell expressing 3-mercaptopyruvate sulfurtransferase (3MST) and cysteine aminotransferase (CAT) but not increased in cells expressing functionally defective mutant enzymes. Sulfurtransferases (EC 184.108.40.206-5) are widely distributed enzyme of prokaryotes and eukaryotes (Nakamura et al..,2000).The enzymes catalyse the transfer of sulfane sulfer from a donor molecules, such as thiosulfate or 3-mercaptopyruvate,to a nucleophilic acceptor, such as cyanide or mercaptoethanol. However, the natural sulfane donors and acceptors and the physiological functions of most sulfurtransferases remain uncertain.
The oxidation of cyanide (CN) to thiocyanate (SCN) is the primary in vivo biochemical pathway for CN detoxification (vennesland et al..,1982).Two enymes, thiosulfate: cyanide sulfurtransferase (rhodanese: E.C. 220.127.116.11) and 3-mercaptopyruvate sulfurtransferase (3-MPST :E.C 18.104.22.168) are considered to be the enzymes primarily responsible for the enzymatic detoxification of CN (Jarabak and Westley ,1978).Rhodanese transfers sulphur from a sulfanessulfur donor molecule to CN (Westley,1981) while 3-MPST catalyses the transfer of sulphur from 3-mercaptopyruvate to CN (Jarabak,1981),both forming SCN (Westley,1981 ;Jarabak ,1981).3-mercaptopyruvate sulfurtransferase (3-MPST:E.C. 22.214.171.124) is an enzyme located in the cytosol and mitochondria of cells and is believed to be involved in the endogenous detoxification of cyanide (CN) because it is capable of transferring sulphur from 3-mercaptopyruvate (3-MP) to CN, forming thiocyanate (SCN).
In addition,3-MPST activity is present in the erythrocytes and cyanide appears to be converted to SCN primarily in the blood, providing further evidence that 3-MPST may make a significant contribution to the endogenous detoxification of cyanide. The major route of metabolism for hydrogen cyanide and cyanides is detoxification in the liver by the mitochondrial cyanide metabolising enzymes; (rhodanese or thiosulphate cyanide/sulphurtransferase: E.C. 126.96.36.199 and mercaptopyruvate sulphurtransferase: E.C. 188.8.131.52) (Barillo, 2009).
SSO + CN SO + SCN
Thiosulphate ion cyanide ion sulphate ion Thiocyanide ion 3-mercaptopyruvate sulfurtransferase function in the detoxifications of cyanide; mediation of sulfur ion transfer to cyanide or to thiol compounds.(Vanden et 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 (Shibuya et 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 condition (Nagahara et al 2005).Hydrogen sulphide (H2S) is an important synaptic modulator, signalling molecule, smooth muscle contractor and neuroprotectant (Hosoki et al 1997).its production by the 3-mercaptopyruvate sulfurtransferase and cysteine aminotransferase pathways is regulated by calcium ions (Hosoki et al 1997). Organisms that are exposed to cyanide poisoning usually have this enzyme in them. This could be in food as in the cyanogenic glucosides 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 is 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)
The rhodanese family sulfurtransfrase are thought to occur in the majority of organisms (bordo and bork, 2002), with the mammalian enzymes being the most extensively studied (Nandi et al.,2002).The first elucidated role of mitochondrial bovine liver rhodanese was the detoxification of cyanide to form thiocyanate, which is harmless and excreted by the kidney. This role could be important, especially in the epithelial cells lining the gut (Picton et al.,2002), but is thought not to account for the wide distribution of this sulfurtransferases in different cells (Nakamura et al.,2002). It has been suggested that CN toxicity could be migitated by enhancing the endogenous cyanide detoxification rate via increasing the availability of sulphur donor molecule, the activity of enzymes involved in CN detoxification( Baskin,2007 ) or both .
Another putative function of at least some sulfurtransferases is the provision of sulfane sulfur required for the formation of the iron-sulfur centers of protein, notably respiratory proteins (Marquet,2001).Sulfurtransferases may also play a part of management of the cytotoxicity of reactive oxygen species in aerobic tissues (Nandi et al.,2002). Most sulfurtransferases have a N-terminal “structural” domain and C-terminal domain containing the active site (Bordo and Bork 2000). The vertebrate rhodaneses have been extensively studied in attempts to understand the part played by the N-terminal structural domain in the correct folding and stability of the enzymes.Bovine rhodanese has a 1000-fold higher affinity for the reduced form of thioredoxin than for cyanide and so many function in peroxide detoxification (Nandi et al .,2002 ). Current evidence suggests, however, that correct protein folding also requires the assistance of a chaperon molecule (Lee and Horowitz,1995).A reaction analogous to that of sulfane-loaded sulfurtransferases with thiouridine (Lauhon and Kambampativ,2000 ; Mueller et al.,2000), and the formation or thiocarboxylate during thiamine biosynthesis by the multidomain protein Thil of Escherichia coli ( Lauhon and Kambampativ,2000 ; Palencer et al., 2000) and molybdopterin (Leimkuhler and Rajagopalan,2001). Moreover, a role for sulfurtransferases in assimilatory sulphate reduction by transferring a molecule of sulfide to O-acetyl-L-serine in the synthesis of cysteine has been postulated (Schmidt and Jager 1992).
Several models have been proposed for the endogenous CN detoxification process, and 3-MPST has a significant role in each of them. The simpliest model proposes that 3-MPST directly transfers sulfur from 3-mercaptopyruvate to CN forming SCN (Sorbo,1957).Alternatively, a recent proposal has suggested that 3-MPST transfers sulfur from3-mercaptopyruvate to albumin in the liver, which then enters the circulation and is available to react with extracellular CN (Westley,1988). 3-MPST activity is high in erythrocytes (Koj et al.,1968)and liver in comparison to rhodanese.
A pharmacokinetic study has determined that the conversion of CN to SCN appears to occur primarily in the blood or tissue areas in proximity to blood. Thus, the result of this pharmacokinetic study is in agreement with either of these two models. Another proposal that has been made suggests that rhodanese and 3-MPST act in concert to detoxify cyanide, with 3-MPST producing sulfane-sulfur pool. These sulfane-sulfur compounds produced by 3-MPST, which may be polysulfides, are subsequently utilized by rhodaneseas sulfur donors. Although the exact role of 3-MPST in the endogenous detoxification process is not known, 3-MPST has a significant role in all of the models proposed for the endogenous CN detoxification process.To develop new sulfur donor foe 3-MPST the factor(s) which determine the substrate specificity of 3-MPST must be determined.
1:1 STRUCTURE OF 3-MERCAPTOPTOPYRUVATE SULFURTRANSFERASE
Leishmania major 3-mercaptopyruvate sulfurtransferase (LmMST) is a crescent-shaped molecule comprising three domains. The N-terminal and central domains are similar to the thiosulfate sulfurtransferase rhodanese and create the active site containing a persulfurated catalytic cysteine (Cys-253) and an inhibitory sulfite coordinated by Arg-74 and Arg-185. A serine protease-like triad, comprising Asp-61, His-75 and Ser-255, is near Cys-253 and represents a conserved feature that distinguishes 3-mercaptopyruvate sulfurtransferases from thiosulfate sulfurtransferases. During catalysis Ser-255 may polarize the carbonyl group of 3-mercaptopyruvate to assist thiophilic attack whilst Arg-74 and Arg-185 bind the carboxylate group. The enzyme hydrolyzes Bz-Arg-pNA, an activity that is sensitive to the presence of the serine protease inhibitor tosyllysylchloromethyl ketone (TLCK). TLCK also lowers 3mercaptopyruvate sulfurtransferase activity, presumably by interference with the contribution of Ser-255. The LmMST is unusual with an 80-amino acid C-terminal domain, bearing remarkable structural similarity to the FK506 binding protein class of peptidyl prolyl cis/trans isomerase. This domain may be involved in mediating protein folding and sulfurtransferase protein interactions.
Sulfurtransferases (EC 184.108.40.206-5) catalyze the transfer of sulfane sulfur from a donor molecule to a thiophilic acceptor. These enzymes are widely distributed in plants, animals and bacteria (Papenbrock, J., and Schmidt, A.,2000) and have been implicated in a wide range of biological processes. For example, sulfurtransferases may be involved in the formation and maintenance of iron-sulfur clusters in protein (Ogata, K., and Volini, M,1990), detoxification of cyanide (Nagahara, N., Ito, T.,1999), degradation of cysteine (Hannestad, U.,1981), biosynthesis of the molybdopterin co-factor of xanthine oxidase (9), selenium metabolism (Nakamura, T.,2000), as well as thiamine and 4-thiouridine biosynthesis (Mueller, E.G., Palenchar, P.M.,2001). The expression of specific sulfurtransferases is up-regulated under conditions of peroxide or hypo-sulfur stress, osmotic shock and phage infection (Adams, H.,2002) suggesting that such enzyme activity is protective of the cell and/or involved in repair processes. Nevertheless, despite intensive study, the biological functions and identification of the physiological substrates of sulfurtransferases remains uncertain. The archetypal sulfurtransferase is rhodanese, a thiosulfate: cyanide sulfurtransferase (TST) able to catalyze the transfer of the thiosulfate sulfur to cyanide in vitro. The related 3-mercaptopyruvate sulfurtransferase (3-mercaptopyruvate: cyanide sulfurtransferase; MST), first discovered in rat liver (Meister, A. 1953), catalyzes similar reactions to rhodanese but uses 3-mercaptopyruvate in preference to thiosulfate as the donor in the two-step reaction :-
HSCH2COCOO- + E ↔ CH2COCOO- + ES [step 1]
ES + CN- ↔ E + SCN- [step 2]
where E represents the free enzyme and ES the enzyme-sulfur adduct.
Crystal structures of rhodaneses have been elucidated and analyzed in detail (Gliubich, F.,1996). The enzyme consists of two domains that, despite a low level of sequence identity, are structurally homologous. Each domain, often referred to as a rhodanese domain, is constructed from a five-stranded β-sheet core surrounded by five α-helical sections. The active site, with a catalytic cysteine, is situated in a cleft formed at the interface of the domains though it is mainly constructed from residues associated with the C-terminal domain. For that reason this domain is often termed the active domain whilst the N-terminal domain is described as inactive.
Although there is no structure yet available for any MST, several observations suggest that they are evolutionarily and structurally related to TSTs (Ogata, K., and Volini, M, 1990). The two types of enzyme catalyze the sulfurtransferase reaction via the formation of a persulfide S -covalently bound to the thiol of a catalytic cysteine, display significant levels of sequence similarity and some are immunologically cross-reactive. However, the different preferred in vitro sulfur donors suggest that the enzymes have different in vivo substrates and physiological roles. Despite the presence of a conserved catalytic cysteine, suggestive of a similar mechanism, the amino acid composition and location of charged residues in the active site of TSTs and MSTs are distinct (Spallarossa,2001). In TSTs, two large and basic residues within the hexapeptide motif Cys-Arg-Lys-Gly-Val-Thr follow the catalytic cysteine. In MSTs, the Arg-Lys pair is replaced by a Gly-Ser or Gly-Thr combination. Mutation of these particular residues to those observed in the other family of enzymes results in partial conversion to that activity, i.e. MST becomes more rhodanese-like and vice versa (Nagahara, N., and Nishino, T. 1996). Studies on the sulfurtransferase SseA, an Escherichia coli protein involved in serine sensitivity, have reinforced the observation that the sequence following the active site cysteine can distinguish sulfurtransferases as TSTs or MSTs (Colnaghi., Cassinelli, ,2001), but the structural consequences of such non-conservative amino acid differences to the active site of MST were unclear.
Williams et al. (Williams ,2002) recently identified and characterized a cytosolic MST from the parasitic trypanosomatid Leishmania major (LmMST). Expression of this enzyme is up regulated in L. major promastigotes during conditions of oxidative stress suggesting an involvement in detoxification of peroxides and, in common with E. coli and mammalian MST (28-30), LmMST is able to utilize thioredoxin as the thiophilic acceptor. It was also reported that LmMST can fold independently (William ,2002) in contrast to many other sulfurtransferases which require molecular chaperones to assist such a process . It was hypothesized that the unusual 80-amino acid C-terminal extension in LmMST may play a part in the folding process, particularly as short truncations of this region resulted in misfolded protein (Williams, 2002).
The availability of a stable and active recombinant enzyme allowed us to initiate a crystallographic study to delineate structure-activity relationships in a MST with the aims of characterizing the active site, investigating the roles of the two residues immediately following the catalytic cysteine, determining the structure of the C-terminal extension, and providing a MST model for detailed comparisons with TSTs. A number of assays using peptidyl-p-nitroanilide (pNA) substrates were carried out seeking to identify additional enzyme activities.
1:2 THE GRASSCUTTER (Thryonomys swinderianus)
Grasscutters are wild herbivorous rodent found in the sub-Saharan region of Africa. They are the biggest after porcupine in the rodent class and mostly referred to as cane rat or cutting grass. Scientifically it is referred to as Thryonomys swinderianus.
Species; Thryonomys swinderianus
The species are of two types, Thryonomys swinderianus which is the giant/greater breed and Thryonomys gregorianus which is the lesser breed. (Rosevear, 1969).
1:2:1 Physical Description of Thryonomys swinderianus
The body length of Thryonomys swinderianus varies from 25-70cm in length (Fitzinger, 1995). The total body weight of adult ranges between 4-12 kg. The body is heavily built, round with bristle fur and coat (Akinloye, 2005).The fur reflects the general colour of the animal, it is brownish from the base to the middle of the fur, while the upper fur appears light yellow. Besides, common colour that ranges from grayish black to brown exists. It is a monogastric herbivore, quick runner, skilled swimmer with poor vision, has a good sense of smell, and lives up to 4years in captivity. It has induced ovulation and a gestation period of 150-156 days with litter size of about six (Opara et al., 2006). They have round nose, short ears and incisors that grow continuously (Mills, 1997). The pelage is coarse, with flattened bristle-like hairs that grow in groups of five or six. The fore feet are smaller than the hind feet. They have five digits, where the three middle digits is well developed, but the first and fifth digits are greatly reduced. The hind feet have no first digit and all digits have heavy claws (Fitzinger, 1995). The dentition for Thryonomys swinderianus is 1/1, 0/0, 1/1, 3/3, representing incisors, canines, premolars and molars respectively (Merwe, 2000). They are endothermic and bilaterally symmetrical. The arrangement of their digits and pads on the fore feet allows a stem to be gripped in one paw only, while being fed into the mouth unlike other rodents that use both paws to hold a single stem (Fitzinger, 1995). Despite their size and short limbs, grasscutters run quickly when disturbed. Their nipples are arranged in lateral position and this makes the young ones to suckle their mother from the side (Asibey, 1974). The head is broad with short flattened muzzle, small eyes and ears (Asibey, 1974).