Rhodanese Human

What is Human Rhodanese and what is its primary function?

Human Rhodanese (Thiosulfate sulfurtransferase, TST) is a mitochondrial matrix enzyme encoded by the nucleus. It primarily functions as a sulfur transferase that catalyzes the detoxification of cyanide through a sulfuration reaction, converting thiosulfate and cyanide to sulfite and thiocyanate . This reaction follows a double displacement (ping-pong) mechanistic pathway, where the enzyme cycles between sulfur-free and sulfur-loaded states . While initially discovered for its role in cyanide detoxification, research has expanded our understanding of rhodanese as a multifunctional enzyme involved in several physiological processes including iron-sulfur protein formation, modification of sulfur-containing enzymes, and participation in sulfur atom trafficking pathways essential for cellular metabolism .

What is the structural composition of Human Rhodanese?

Human Rhodanese is a single polypeptide chain containing approximately 297 amino acids with a molecular weight of around 35.6-37 kDa . The enzyme features two highly conservative domains identified as rhodanese homology domains . The active site contains a critical tryptophanyl residue positioned in close proximity to an essential sulfhydryl group, which facilitates the enzyme's catalytic activity . The "rhodanese fold" represents a specific α/β structural arrangement containing approximately 100 residues that is found across diverse protein structures . This structural motif is characterized by conserved elements that support the enzyme's function in sulfur transfer reactions while maintaining the conformational stability required for catalysis under physiological conditions .

How is Rhodanese activity measured in experimental settings?

The most widely accepted method for measuring rhodanese activity is based on the colorimetric estimation of thiocyanate formed from the reaction of cyanide and thiosulfate catalyzed by the enzyme . In this assay, the reaction mixture typically contains the enzyme sample, sodium thiosulfate as the sulfur donor, and potassium cyanide as the acceptor in an appropriate buffer system. After incubation, the reaction is stopped, and the thiocyanate produced is quantified spectrophotometrically using ferric nitrate reagent, which forms a colored complex with thiocyanate . The intensity of the resulting color is proportional to the amount of thiocyanate formed, reflecting the enzyme's activity. This method allows researchers to express activity in terms of μmol of thiocyanate formed per minute under standardized conditions, enabling comparative analysis across different experimental setups and biological samples.

What factors influence Rhodanese activity in biological systems?

Rhodanese activity exhibits significant variability based on several biological and environmental factors. Research has demonstrated that activity levels are modulated by species differences, organ specificity, sex, age, and dietary influences . Within different tissues, rhodanese shows varied distribution patterns, with highest activities typically observed in liver and kidney tissues that play critical roles in detoxification processes . Environmental factors such as exposure to cyanogenic compounds can induce adaptive responses in rhodanese expression and activity . At the molecular level, post-translational modifications, protein-protein interactions, and the availability of substrate pools (particularly sulfur donors) significantly impact the enzyme's catalytic efficiency . In experimental settings, factors such as pH, temperature, ionic strength, and the presence of specific activators or inhibitors must be carefully controlled to obtain reproducible activity measurements that reflect the physiological state of the enzyme.

How does the dual-domain structure of Rhodanese contribute to its catalytic versatility?

The rhodanese fold is a ubiquitous structural arrangement found in various protein subfamilies associated with different physiological functions . Human rhodanese contains two domains with similar folding patterns but different functional properties. The C-terminal domain contains the active site cysteine residue critical for catalysis, while the N-terminal domain provides structural support and may facilitate substrate binding and product release . This domain architecture enables rhodanese to participate in multiple catalytic activities beyond the classical thiosulfate:cyanide sulfurtransferase reaction. Recent research has revealed that rhodanese domains can catalyze phosphatase reactions linked to cell cycle regulation and may be involved in tRNA hydroxylation, illustrating the catalytic versatility conferred by this dual-domain structure . The evolutionary conservation of this fold across diverse protein families suggests that the domain arrangement provides a versatile scaffold for different nucleophilic reactions, particularly those involving sulfur chemistry, enabling rhodanese-containing proteins to participate in numerous biochemical pathways including sulfur trafficking, hydrogen sulfide metabolism, biosynthesis of molybdenum cofactor, and thio-modification of tRNAs .

What is the mechanism of selenium substitution in Rhodanese and its functional implications?

The active site cysteine of rhodanese can be substituted with selenium under specific conditions, forming a selenium-substituted enzyme (E-Se rhodanese) . This substitution occurs when persulfide-free bovine-liver rhodanese (E form) reacts with selenium compounds in the presence of glutathione (GSH) . The formation of E-Se rhodanese is most effective when the ratio of GSH:SeO₃²⁻ is 4:1, resulting in the binding of one molar equivalent of selenium per mole of enzyme . The reaction requires an intermediate produced from the interaction of selenite with GSH, likely the selenotrisulfide GSSeSG .

The selenium-substituted enzyme exhibits distinct stability characteristics compared to low molecular weight selenium compounds; while GSSeSG readily decomposes to oxidized GSH and elemental selenium, E-Se rhodanese remains stable even after extended incubation . This stability suggests a potential biological role for rhodanese in selenium metabolism. Critical to this substitution is the availability of the free active-site cysteine, as carboxymethylated rhodanese or sulfur-loaded rhodanese cannot bind selenium . The functional implications of this selenium substitution may include altered catalytic properties, potential roles in selenium trafficking, and possible protective effects against selenium toxicity, though these aspects require further investigation.

How does Rhodanese contribute to prevention of hyperglycemic damage in diabetic conditions?

Recent research has identified thiosulfate sulfurtransferase (TST/Rhodanese) as a genetic predictor of resistance to obesity-related type 2 diabetes, suggesting a previously unrecognized role in glucose metabolism . In diabetic conditions characterized by hyperglycemia, rhodanese appears to exert protective effects against tissue damage. Studies using zebrafish models demonstrated that incubation with human TST (hTST) and thiosulfate rescued the hyperglycemic phenotype in pdx1-knockdown zebrafish pronephros .

The protective mechanism likely involves the activation of the mitochondrial TST pathway, which may counteract the oxidative stress and metabolic dysregulation associated with hyperglycemia . This function could be linked to rhodanese's ability to participate in sulfur metabolism and potentially hydrogen sulfide (H₂S) production, as H₂S has been identified as a gasotransmitter with significant physiological roles including cytoprotection and anti-inflammatory effects . The finding that rhodanese can prevent hyperglycemic damage suggests it may represent a promising target for therapeutic intervention in diabetes and its organ complications, particularly diabetic nephropathy and retinopathy . This emerging role expands our understanding of rhodanese beyond its classical detoxification function, highlighting its significance in metabolic homeostasis.

What role does Rhodanese play in the integrated network of cellular sulfur trafficking?

Rhodanese functions as a key node in the complex network of cellular sulfur trafficking, participating in multiple pathways that maintain sulfur homeostasis . This role extends beyond simple cyanide detoxification to encompass essential processes such as:

• Transfer of sulfur atoms for iron-sulfur cluster assembly in mitochondrial proteins

• Contribution to hydrogen sulfide (H₂S) metabolism, supporting its function as a gasotransmitter

• Involvement in the biosynthesis of molybdenum cofactor, essential for several oxidoreductases

• Participation in thio-modification of tRNAs, affecting translation fidelity and efficiency

• Potential roles in protein urmylation, a post-translational modification pathway

The enzyme's catalytic mechanism enables it to accept sulfur from various donors (primarily thiosulfate) and transfer it to different acceptors, functioning as a sulfur carrier in cellular compartments . This versatility allows rhodanese to integrate with multiple biochemical pathways, responding to changing cellular needs for sulfur-containing compounds and detoxification requirements. The importance of this integration is highlighted by the evolutionary conservation of rhodanese domains across species and their presence in multiple protein subfamilies with diverse physiological functions . Understanding how rhodanese coordinates these various sulfur trafficking pathways remains an active area of research with implications for numerous physiological processes and disease states.

What are the optimal conditions for expressing and purifying recombinant Human Rhodanese?

The expression and purification of recombinant human rhodanese requires careful optimization of conditions to ensure proper folding and enzymatic activity. Based on established protocols, the following methodological approach has proven effective:

For optimal activity preservation, it's crucial to avoid multiple freeze-thaw cycles and to maintain protein concentration above 1 mg/ml . The purified enzyme should demonstrate >95% purity as determined by SDS-PAGE and maintain its characteristic sulfurtransferase activity . For functional studies, it's important to ensure the active site cysteine remains in a reduced state, as oxidation can significantly diminish catalytic activity. This can be achieved by including reducing agents such as DTT or β-mercaptoethanol during purification steps and storage.

How can researchers effectively measure rhodanese enzyme kinetics?

Measuring rhodanese enzyme kinetics requires careful experimental design to account for the ping-pong bi-substrate reaction mechanism. The following methodology provides a robust approach:

• Reaction setup: Standard assay conditions include 50 mM potassium phosphate buffer (pH 7.4), sodium thiosulfate (1-50 mM range for Km determination), potassium cyanide (1-50 mM range), and purified enzyme (1-5 μg) .

• Kinetic measurements: For accurate determination of kinetic parameters:

  • Hold one substrate constant at saturating concentration while varying the other

  • Measure initial reaction rates across a range of substrate concentrations

  • Plot data using Lineweaver-Burk or Eadie-Hofstee transformations for ping-pong mechanisms

• Data analysis: For the ping-pong bi-substrate mechanism, the rate equation is:
  $v = \frac{V_{max}[A][B]}{K_m^B[A] + K_m^A[B] + [A][B]}$
  where [A] and [B] are substrate concentrations and K_m^A and K_m^B are the respective Michaelis constants.

• Controls and considerations:

  • Include enzyme-free controls to account for non-enzymatic reactions

  • Monitor potential substrate inhibition at high concentrations

  • Consider the influence of pH and temperature on kinetic parameters

  • Ensure linearity of the assay with respect to time and enzyme concentration

This approach allows for accurate determination of kinetic parameters including K_m, k_cat, and catalytic efficiency (k_cat/K_m) for both substrates, providing insight into the catalytic mechanism and potential effects of modulators on enzyme activity.

What approaches can be used to investigate the interaction between Rhodanese and potential protein partners?

Investigating protein-protein interactions involving rhodanese requires multiple complementary approaches to establish physiologically relevant associations. The following methodological strategies are recommended:

• Co-immunoprecipitation (Co-IP):

  • Use antibodies against rhodanese or the target protein to pull down protein complexes from cell lysates

  • Western blot analysis of the precipitated complexes can confirm the interaction

  • Controls should include IgG-only precipitations and lysates from cells where rhodanese is knocked down

• Proximity-based labeling techniques:

  • BioID or TurboID approaches fuse rhodanese to a biotin ligase that biotinylates nearby proteins

  • APEX2 fusion creates an electron microscopy-visible tag and enables proximity biotinylation

  • These methods can identify transiently interacting proteins in the native cellular environment

• Förster Resonance Energy Transfer (FRET):

  • Tag rhodanese and potential partners with appropriate fluorophores

  • Measure energy transfer as an indicator of protein proximity (<10 nm)

  • Particularly useful for monitoring dynamic interactions in living cells

• Surface Plasmon Resonance (SPR):

  • Immobilize purified rhodanese on a sensor chip

  • Measure binding kinetics (kon, koff) and affinity (KD) of potential partners

  • Provides quantitative data on interaction strength and dynamics

• Crosslinking mass spectrometry (XL-MS):

  • Use chemical crosslinkers to stabilize protein complexes

  • Digest and analyze by mass spectrometry to identify interaction interfaces

  • Provides structural insights into the topology of the protein complex

What methods are effective for studying the role of Rhodanese in cyanide detoxification pathways?

Studying rhodanese's role in cyanide detoxification requires approaches that span from molecular mechanisms to physiological outcomes. The following methodological framework provides comprehensive analysis:

• In vitro enzymatic assays:

  • Measure thiocyanate formation using colorimetric methods with ferric nitrate reagent

  • Determine enzyme kinetics with varying concentrations of cyanide and thiosulfate

  • Assess the effect of potential inhibitors or activators on detoxification efficiency

• Cellular models of cyanide exposure:

  • Culture relevant cell types (hepatocytes, kidney cells) with controlled cyanide concentrations

  • Manipulate rhodanese levels through overexpression or knockdown approaches

  • Measure cell viability, mitochondrial function, and detoxification capacity

• Metabolic flux analysis:

  • Use isotopically labeled cyanide (e.g., ¹³C or ¹⁵N) to track detoxification pathways

  • Measure labeled thiocyanate formation using mass spectrometry

  • Determine the relative contribution of rhodanese versus other detoxification pathways

• Animal models for in vivo assessment:

  • Utilize rhodanese knockout/knockdown or overexpression models

  • Challenge with sub-lethal cyanide doses and monitor physiological responses

  • Measure tissue-specific enzyme activities and correlate with detoxification capacity

• Structural biology approaches:

  • Perform site-directed mutagenesis of key active site residues

  • Analyze crystal structures of enzyme-substrate complexes

  • Use molecular dynamics simulations to understand reaction mechanisms

This multi-level approach provides a comprehensive understanding of rhodanese's contribution to cyanide detoxification, from molecular mechanisms to physiological significance, while accounting for potential compensatory pathways and tissue-specific responses.

How might rhodanese be targeted for therapeutic applications in metabolic disorders?

Recent discoveries regarding rhodanese's role in metabolic homeostasis, particularly its potential protective effects against hyperglycemic damage, open new avenues for therapeutic applications . The enzyme has been identified as a genetic predictor of resistance to obesity-related type 2 diabetes, suggesting it may serve as a novel target for metabolic interventions . Several approaches show promise for therapeutic development:

• Activation of the mitochondrial TST pathway: Research in zebrafish models demonstrated that administration of human TST protein along with thiosulfate rescued hyperglycemic phenotypes in diabetic models . This suggests that enhancing rhodanese activity could potentially mitigate diabetic complications in tissues such as kidneys and retina.

• Modulation of hydrogen sulfide metabolism: Rhodanese's involvement in sulfur trafficking may influence H₂S levels, which have beneficial effects on vascular function, inflammation, and insulin sensitivity . Therapies targeting this pathway could enhance H₂S signaling in metabolic disorders.

• Development of small molecule modulators: Structure-based drug design could yield compounds that enhance rhodanese activity or stability in specific tissues, potentially providing tissue-targeted approaches to managing metabolic dysfunction.

• Gene therapy approaches: For genetic disorders affecting rhodanese function, targeted gene therapy to restore proper expression levels could be explored, particularly in tissues where the enzyme plays critical protective roles.

The development of these therapeutic approaches requires further research to fully elucidate rhodanese's tissue-specific functions, identify optimal administration routes and formulations, and understand potential off-target effects. Clinical translation will depend on establishing clear biomarkers of rhodanese activity and developing methods to monitor therapeutic efficacy in metabolic disorder patients.

What is the potential role of Rhodanese in cancer biology and possible therapeutic applications?

The relationship between rhodanese and cancer biology remains an emerging area of research with potential therapeutic implications. Several aspects of rhodanese function intersect with cancer-related processes:

• Redox homeostasis regulation: Rhodanese's involvement in sulfur metabolism impacts cellular redox balance, which is often dysregulated in cancer cells . This connection suggests potential roles in tumor progression or suppression depending on the cancer type and microenvironment.

• Mitochondrial function: As a mitochondrial enzyme, rhodanese may influence mitochondrial metabolism and integrity, which are increasingly recognized as important factors in cancer development and therapeutic response .

• Detoxification capacity: Cancer cells often exhibit altered detoxification mechanisms. Changes in rhodanese expression or activity could affect cellular responses to both endogenous toxins and chemotherapeutic agents .

• Potential applications in cancer therapy:

  • Rhodanese could potentially be exploited for cancer-specific activation of prodrugs containing cyanide or other groups that can be metabolized by the enzyme

  • Differences in expression between normal and cancer tissues might provide opportunities for targeted interventions

  • The enzyme's role in sulfur metabolism could be targeted to disrupt cancer-specific metabolic adaptations

Further research is needed to characterize rhodanese expression patterns across cancer types, understand how its function impacts tumor biology, and develop potential therapeutic strategies based on these insights. The integration of rhodanese-focused studies with broader cancer metabolism research may reveal novel approaches to cancer diagnosis, prognosis, and treatment.