Fumarase Human

What is the basic structure of human fumarase and how does it relate to its function?

Human fumarase is a homotetramer composed of four identical subunits, each containing three domains (D1, D2, and D3). The enzyme has a molecular mass of approximately 53.8 kDa per monomer . The quaternary structure is essential for full enzymatic activity since each active site requires residues from three of the four subunits within the homotetramer .

The D2 domain facilitates tetramerization, forming the major intersubunit interface upon oligomerization . The D1 and D3 domains lie at the corners of the homotetramer and outline the entry points to the four independent active sites . The active sites are located between domains D1 and D3 at the four corners of the enzyme .

Three regions within the fumarase family show high conservation:

• Region 1 (residues 176-193)

• Region 2 (residues 228-247)

• Region 3 (residues 359-381, also known as the "signature sequence")

Regions 1 and 3 form the majority of the substrate-binding site, while regions 2 and 3 contribute the catalytic groups H235 and S365 .

What is the dual localization of human fumarase and its significance in cellular function?

Research has revealed that cytosolic fumarase plays a key role in protecting cells from DNA damage, particularly from DNA double-strand breaks . Upon DNA damage induction, cytosolic fumarase is recruited to the nucleus as part of the DNA damage response . This function depends on its enzymatic activity and can be complemented by high concentrations of fumaric acid .

This dual localization suggests an intriguing crosstalk between primary metabolism and the DNA damage response, potentially explaining the tumor suppressor role of fumarase in human cells .

What are the optimal conditions for measuring human fumarase activity in vitro?

Human fumarase activity can be measured using several approaches:

Direct Spectrophotometric Assay:

• The formation of fumarate from L-malate can be monitored by measuring the increase in absorbance at 240 nm due to the alpha-beta unsaturated bond in fumarate .

• Standard assay conditions typically use 100 mM potassium phosphate buffer at pH 7.6 .

Coupled Enzyme Assay:

• A fluorescence-based coupled assay can be used, where the FH reaction is linked to malate dehydrogenase (MDH) and diaphorase reactions .

• In this format, L-malate produced by FH is converted to oxaloacetate by MDH, generating NADH.

• Diaphorase then uses NADH to convert resazurin to fluorescent resorufin, which can be measured at excitation 530-540 nm and emission 585-595 nm .

For accurate kinetic measurements, it's essential to consider the reversible nature of the fumarase reaction. The contribution of both forward and reverse reactions should be analyzed simultaneously for precise determination of kinetic parameters .

How can recombinant human fumarase be expressed and purified for biochemical studies?

Expression and purification of recombinant human fumarase typically involves:

• Construct Design: Using an E. coli codon-optimized human fumarase domain (residues 44-510) with an N-terminal His6-tag .

• Expression System: Expression in E. coli has been demonstrated to produce functional enzyme, indicating that human fumarase does not require human-specific post-translational modifications for basic enzymatic function .

• Purification Process:

  • Affinity chromatography using Ni-NTA resin for His-tagged protein

  • Size exclusion chromatography can be used to separate tetrameric active enzyme from other oligomeric forms

• Verification: SDS-PAGE typically shows a band at approximately 53.8 kDa, confirming the expected molecular mass .

The purified recombinant human fumarase follows Michaelis-Menten kinetic behavior with a Vmax of approximately 170 μmols/min/mg enzyme and a Km for L-malate of about 1.9 mM .

What methods are available for assessing the oligomerization state of human fumarase?

Several complementary methods can be used to analyze the oligomerization state of human fumarase:

• Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE):

  • Allows visualization of native protein complexes without denaturing them

  • Can distinguish between tetrameric, dimeric, and monomeric forms

• Gel Filtration Chromatography:

  • Separates proteins based on their hydrodynamic volume

  • Can be used to determine approximate molecular weights of native complexes

  • Effective for comparing wild-type tetrameric fumarase versus variant forms with altered oligomerization

• Differential Scanning Fluorimetry (DSF):

  • Measures thermal stability of proteins

  • Can detect differences in stability between properly oligomerized and improperly assembled variants

• X-ray Crystallography:

  • Provides high-resolution structural information about quaternary structure

  • Has been used to solve the human fumarase structure at 1.8 Å resolution

What are the known disease-causing mutations in the FH gene and their biochemical consequences?

Several mutations in the FH gene have been associated with disease, particularly Fumarase Deficiency and Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC). Some well-characterized mutations include:

These mutations typically impact the enzyme's quaternary structure by disrupting the intersubunit interface within the D2 domain . Since the active site requires contributions from three of the four subunits, defective oligomerization directly impairs enzymatic activity .

The A308T and H318Y variants show severely diminished fumarase activity primarily due to decreased turnover rate (kcat), while maintaining Km values for L-malate similar to the wild-type enzyme .

How does fumarase deficiency manifest clinically and what are the diagnostic approaches?

Fumarase deficiency primarily affects the nervous system, especially the brain. Clinical manifestations include:

• Abnormally small head size (microcephaly)

• Abnormal brain structure

• Severe developmental delay

• Weak muscle tone (hypotonia)

• Failure to gain weight and grow at the expected rate (failure to thrive)

• Seizures

• Distinctive facial features (prominent forehead, low-set ears, small jaw, widely spaced eyes, depressed nasal bridge)

• Hepatosplenomegaly (enlarged liver and spleen)

• Hematological abnormalities (polycythemia or leukopenia)

Most affected individuals survive only a few months, but some have lived into early adulthood .

Diagnostic approaches include:

• Enzymatic Assays: Measuring fumarase activity in various tissues, including white blood cells, fibroblasts, or muscle biopsies

• Metabolic Screening: Detection of elevated fumarate in urine or blood

• Genetic Testing: Sequencing of the FH gene to identify pathogenic mutations

Fumarase deficiency is inherited in an autosomal recessive pattern, requiring mutations in both copies of the FH gene .

How does fumarase function in the DNA damage response pathway?

Studies have revealed that cytosolic fumarase plays a critical role in the DNA damage response (DDR) . Key aspects of this function include:

• Nuclear Translocation: Upon DNA damage induction, cytosolic fumarase is recruited from the cytosol to the nucleus .

• Enzymatic Activity Requirement: This function depends on fumarase's enzymatic activity, as the absence of the enzyme can be complemented by high concentrations of fumaric acid .

• Metabolic-DDR Crosstalk: This represents an exciting interaction between primary metabolism and the DNA damage response, suggesting metabolic control of tumor propagation .

• HIF Independence: The DNA damage response function of fumarase is most likely independent of the hypoxia-inducible factor (HIF) pathway that was previously suggested as a mechanism for HLRCC tumorigenesis .

• Contradictory Cellular Function: Fumarase has a contradictory cellular function - it is pro-survival through its role in the TCA cycle, yet its loss can drive tumorigenesis in certain contexts .

This dual role is still being actively investigated to fully understand the molecular mechanisms by which cells adapt to fumarase loss while maintaining proliferative capacity.

What are the current methods to study the adaptive mutations arising from fumarase loss in cells?

Cells that survive fumarase loss develop adaptive mutations to overcome metabolic constraints. Current methods to study these adaptations include:

• CRISPR-Cas9 Knockout Systems: Generation of FH knockout cell lines enables study of cellular responses before and after adaptation .

• Temporal Analysis: Examining cells at different time points after FH loss reveals that the cellular response occurs in two distinct phases:

  • Early phase: Inhibited proliferation and DNA damage repair

  • Late phase: Recovery of proliferation through adaptive mutations

• Whole-Exome Sequencing: Systematic identification of adaptive mutations in FH-knockout clones reveals recurring mutations in oncogenic signaling pathways like JAK/STAT .

• Functional Validation: Testing whether identified mutations are responsible for restoring proliferation under TCA cycle malfunction .

• Metabolomic Analysis: Measuring metabolite levels to understand how cells adapt their metabolism to overcome fumarase loss.

This research is significant for understanding mechanisms of tumor development in HLRCC and potentially identifying therapeutic vulnerabilities.

What approaches can be used to identify modulators of human fumarase activity?

Several approaches can be used to identify compounds that modulate fumarase activity:

• Fluorescence-based Coupled Assays: High-throughput screening can be performed using a coupled enzyme assay system where fumarase activity is linked to malate dehydrogenase and diaphorase reactions, with readout through fluorescent resorufin .

• Structure-Based Drug Design: The 1.8 Å resolution crystal structure of human fumarase provides a basis for in silico screening of potential modulators .

• Allosteric Site Targeting: The binding site for HEPES at the C-terminal domain (Domain 3) involving Lys467 represents a potential allosteric regulation site that could be targeted for modulation .

• Differential Scanning Fluorimetry (DSF): This technique can identify compounds that stabilize or destabilize the enzyme, potentially affecting its activity .

• Reversible Reaction Analysis: Comprehensive kinetic analysis incorporating both forward and reverse reactions simultaneously provides more accurate assessment of how compounds affect enzyme function .

These approaches are valuable for both basic research and potential therapeutic development for conditions associated with altered fumarase activity.

Biochemical Characterization and Parameters

Various buffer conditions and additives can significantly impact human fumarase stability and activity:

• Buffer Systems:

  • 100 mM potassium phosphate buffer at pH 7.6 is commonly used for activity assays

  • HEPES buffer has been found to interact with human fumarase at the C-terminal domain (Domain 3), potentially affecting allosteric regulation

  • Human fumarase catalytic efficiency is higher in the presence of HEPES

• Stability Factors:

  • Urea at concentrations of 1-4 M can reduce enzyme stability over time

  • DTT (dithiothreitol) at 2 mM can help maintain stability, even in the presence of denaturing agents like urea

  • DTNB (5,5'-dithiobis-(2-nitrobenzoic acid)) can affect enzyme stability through mixed-disulfide formation with thiol groups

• pH Effects:

  • pH can influence both stability and activity, with pH 7.6 being optimal for many assay conditions

  • Different pH conditions (e.g., pH 8.5) may affect thiol reactivity and disulfide formation

• Temperature Sensitivity:

  • Differential scanning fluorimetry (DSF) has been used to assess thermal stability of the enzyme

  • Proper tetramerization contributes to thermal stability, with variants showing altered oligomerization typically having reduced stability

Understanding these factors is crucial for experimental design, particularly when studying variants or screening for modulators of enzyme activity.