MAT2A Human

What is human MAT2A and what is its primary function?

Human MAT2A (Methionine Adenosyltransferase 2A) is a member of the AdoMet synthase family, serving as an essential magnesium- and potassium-dependent enzyme that catalyzes the formation of S-adenosylmethionine (SAM or SAMe) . This 395 amino acid isoform functions primarily in extrahepatic tissues, unlike its counterpart MAT1A which is exclusively expressed in the liver . The primary function of MAT2A is to produce SAM, which serves as the principal methyl donor for most biological transmethylation reactions including methylation of nucleic acids, phospholipids, histones, biologic amines, and proteins . MAT2A is found in both the cytosol and nucleus, where it can also function as a transcriptional regulator beyond its enzymatic role .

How does MAT2A differ from MAT1A in terms of structure and tissue distribution?

Despite sharing 84% primary sequence identity and 93% sequence similarity with nearly identical structures (RMSD <1 Å), MAT1A and MAT2A exhibit differential expression patterns . MAT1A is expressed exclusively in the liver where approximately 85% of methylation reactions and the bulk of methionine metabolism occur . In contrast, MAT2A is more widely expressed throughout the body and serves as the main source of SAM outside the liver, including in cancer cells . This distinct tissue distribution suggests specialized roles despite their structural similarities, with MAT2A likely adapting to unique metabolic needs of various extrahepatic tissues .

What is the quaternary structure of active MAT2A?

MAT2A primarily exists in an active dimeric conformation composed of two monomeric subunits . This dimeric structure is functionally significant as the two subunits contribute residues to form two active sites at the subunit interface . The active enzyme contains binding sites for both substrates (ATP and L-methionine) as well as the necessary metal cofactors (magnesium and potassium) required for catalytic activity . Understanding this quaternary structure is essential for interpreting enzyme kinetics and designing potential inhibitors, as the active sites are formed at the interface between the two subunits rather than being contained wholly within individual monomers .

What is the kinetic mechanism of human MAT2A?

Human MAT2A follows a strictly ordered kinetic mechanism based on comprehensive kinetic studies including initial velocity patterns, product inhibition studies, and dead-end inhibition analyses . In this mechanism, ATP binds to the enzyme first, followed by L-methionine binding . After the catalytic reaction occurs, the products are released in a specific order: SAM is released first, followed by random release of phosphate (Pi) and pyrophosphate (PPi) . This ordered mechanism has been confirmed through isothermal titration calorimetry (ITC) experiments, which demonstrated binding of ATP to MAT2A with a Kd of 80 ± 30 μM (close to the Km(ATP) of 50 ± 10 μM), while L-methionine showed no binding to MAT2A in the absence of ATP . This finding resolves previous conflicting reports about the kinetic mechanism of MAT2A that had proposed various sequential ordered and random mechanisms .

What are the key kinetic parameters of human MAT2A?

The key kinetic parameters of human MAT2A have been determined through multiple independent experiments and are presented in the following table:

These values were determined at pH 7.5 and 22°C using a coupled phosphate detection assay . The relatively slow turnover number (kcat = 0.34 s-1) is consistent with values reported by other groups using different assay methods (0.27 and 0.35 s-1) . This slow catalytic rate may be particularly significant in rapidly dividing cells such as cancer cells, where the demand for SAM might be heightened . The Km values for both substrates indicate relatively high affinity, particularly for L-methionine .

How is the catalytic reaction of MAT2A thought to proceed at the molecular level?

The catalytic reaction of MAT2A proceeds through a SN2 transition state for SAM formation . Upon substrate binding, the first step of the reaction occurs when the sulfur atom of L-methionine performs a nucleophilic attack on the 5′-carbon of ATP, forming the product SAM and the intermediate tripolyphosphate (PPPi) . The second step in the reaction is the hydrolysis of enzyme-bound PPPi to produce PPi and Pi as products . This two-step mechanism requires proper orientation of both substrates in the active site, which explains the ordered binding mechanism where ATP must bind first to prepare the active site for L-methionine binding . Understanding this detailed mechanism is crucial for developing potential inhibitors that might target specific steps in the catalytic process.

How does MAT2B interact with MAT2A and what is the functional significance?

MAT2B interacts with MAT2A with high affinity, exhibiting a Kd of 6 ± 1 nM and a binding stoichiometry of 2:1 (MAT2A/MAT2B) . Despite this tight binding, isothermal titration calorimetry and kinetic studies have shown that under conditions where all MAT2A is expected to be in complex with MAT2B, the steady-state kinetic parameters are indistinguishable from those of MAT2A alone . The primary effect of MAT2B appears to be stabilization of MAT2A, particularly at low concentrations (<100 nM) where MAT2A rapidly loses activity at 37°C . In the presence of MAT2B at the known 2:1 MAT2A/MAT2B stoichiometry, MAT2A retained full activity for at least 2 hours . This stabilizing effect may have been misinterpreted in previous studies as activation of MAT2A, particularly in experiments that included preincubation steps without recognizing the inherent instability of MAT2A under those conditions .

What cellular mechanisms regulate MAT2A expression and activity?

Recent research has implicated the RNA binding protein and methyltransferase METTL16 in regulating cellular MAT2A concentrations in response to SAM levels . When SAM concentrations are high, METTL16 (which utilizes SAM as a substrate) binds and methylates MAT2A mRNA, resulting in intron retention and degradation . This causes a reduction in cellular concentrations of the MAT2A protein and consequently SAM . Conversely, when cellular SAM concentrations are low, methylation of MAT2A mRNA by METTL16 decreases, resulting in increased levels of MAT2A mRNA being translated into protein and a subsequent increase in cellular SAM concentration . This feedback mechanism allows METTL16 to act as a SAM sensor that directly regulates the amount of MAT2A enzyme in the cell, maintaining SAM homeostasis . This regulatory mechanism represents a more significant control point for cellular SAM levels than the modest effects of MAT2B on MAT2A activity reported in some studies .

What recombinant protein constructs are typically used for studying human MAT2A?

For studying human MAT2A, researchers typically use recombinant constructs containing the full-length human MAT2A cDNA . In published studies, plasmids encoding human MAT2A have been obtained from repositories such as the Structural Genomics Consortium (SGC) Oxford, with the recombinant construct containing the full-length human MAT2A (UniProt P31153) cloned into vectors such as pNIC28-Bsa4 (GenBank: EF198106.1) . These constructs typically include additional amino acids at the N-terminus containing a His6 tag and a tobacco etch virus protease (TEVp) cleavage site to facilitate purification . The expression construct for human MAT2B (UniProt Q9NZL9) similarly contains the full-length human MAT2B V2 (GenBank: AAH05218.1) with an additional N-terminal sequence containing a His6 tag and TEVp cleavage site . These constructs allow for the production of pure protein suitable for detailed kinetic and structural studies.

What assays are recommended for measuring MAT2A activity and inhibition?

Several complementary assays are recommended for comprehensive characterization of MAT2A activity and inhibition. Coupled phosphate detection assays have been successfully used to determine steady-state kinetic parameters (kcat, Km(ATP), Km(L-Met), and Ki(ATP)) . Additionally, LC-MS SAM detection assays provide direct measurement of the SAM product formation . For analyzing the binding mechanism, isothermal titration calorimetry (ITC) is valuable for determining dissociation constants (Kd) for substrate binding to MAT2A and for protein-protein interactions like MAT2A-MAT2B . When studying inhibition, both product inhibition (using SAM, phosphate, and pyrophosphate) and dead-end inhibition (using L-Met analogs like cycloleucine) approaches provide insights into the mechanism of inhibition . For researchers specifically interested in MAT2A stability, activity assays following preincubation at 37°C can reveal the stabilizing effect of binding partners like MAT2B . The integration of multiple assay types provides a more complete understanding of MAT2A function and the effects of potential inhibitors.

What are the key considerations when designing inhibitor studies for MAT2A?

When designing inhibitor studies for MAT2A, researchers should consider several critical factors based on the enzyme's unique properties. First, understanding that MAT2A follows an ordered kinetic mechanism where ATP binds before L-methionine is essential for interpreting inhibition patterns and designing competitive inhibitors against either substrate . Second, researchers should be aware that MAT2A is unstable at low concentrations (<100 nM), rapidly losing activity at 37°C, which can complicate inhibition studies if not properly controlled . Third, since MAT2B binds at the same allosteric site on MAT2A as all known MAT2A inhibitors (including compounds in human clinical trials), potential allosteric inhibitors could compete with MAT2B in the cellular environment, potentially decreasing their cellular potency and efficacy . Additionally, assay design should account for the relatively slow turnover number of MAT2A (kcat = 0.34 s-1), which may require longer incubation times to observe significant activity . Finally, researchers should consider employing multiple complementary assay methods to fully characterize inhibitor mechanisms, including both functional assays that measure catalytic activity and binding assays to determine direct interactions with the enzyme.

Why is MAT2A considered a potential anti-cancer target?

MAT2A is considered a potential anti-cancer target because it is the primary source of SAM in cancer cells, where methylation reactions play crucial roles in altered cellular processes . Unlike MAT1A which is expressed exclusively in the liver, MAT2A is expressed in various tissues including cancer cells and tumors . The ubiquitous expression of MAT2A outside the liver combined with its critical role in producing SAM makes it a pharmacologically validated anti-cancer target . Interest in MAT2A as a therapeutic target has led to the development of inhibitors such as AG-270, which has entered human clinical trials . The relatively slow turnover rate of MAT2A (kcat = 0.34 s-1) might be particularly significant in rapidly dividing cancer cells where the demand for SAM is high, potentially making inhibition of this enzyme an effective strategy for reducing cancer cell proliferation .

How does MAT2A differ in normal versus cancer cells?

While the search results do not provide specific information about differences in MAT2A between normal and cancer cells, several inferences can be made based on the provided information. MAT2A is expressed in various tissues outside the liver, including cancer cells . The enzyme's role in producing SAM, which is critical for numerous methylation reactions, suggests that rapidly dividing cancer cells with high metabolic demands may have altered MAT2A expression or activity to meet their enhanced needs for methylation reactions . The fact that MAT2A has been validated as an anti-cancer target and that inhibitors like AG-270 have entered clinical trials indicates that there are likely cancer-specific vulnerabilities related to MAT2A function . Additionally, the RNA binding protein METTL16 regulates cellular MAT2A concentrations in response to SAM levels, suggesting that dysregulation of this control mechanism might occur in cancer cells to maintain elevated SAM production . Further research is needed to fully characterize the specific differences in MAT2A expression, regulation, and function between normal and cancer cells.

What approaches have been used to develop MAT2A inhibitors?

The development of MAT2A inhibitors has benefited from detailed understanding of the enzyme's structure and kinetic mechanism. Knowledge that MAT2A follows an ordered kinetic mechanism where ATP binds first, followed by L-methionine, has guided the design of assays capable of identifying novel inhibitors . Both enzymatic and binding assays have been utilized to identify and characterize potential inhibitors . The observation that MAT2B binds at the same allosteric site on MAT2A as all known MAT2A inhibitors is significant for inhibitor development, as it suggests that allosteric regulation is a viable approach . Compounds such as AG-270 have been developed as MAT2A inhibitors and have progressed to human clinical trials . The design of these inhibitors likely considers the competition with MAT2B in the cellular environment, as allosteric inhibitors could compete with MAT2B, potentially affecting their cellular potency and efficacy . Understanding product inhibition by SAM and dead-end inhibition by L-methionine analogs like cycloleucine has also contributed to inhibitor development strategies .

What are the key unresolved questions about MAT2A function and regulation?

Several important questions about MAT2A function and regulation remain unresolved. First, while MAT2B has been characterized as a binding partner of MAT2A, its true physiological role in regulating MAT2A activity remains unclear given the conflicting reports and the findings that it primarily stabilizes rather than significantly alters MAT2A's catalytic activity . Second, the absolute concentration of MAT2A (and MAT2B) in different cell types under various physiological conditions needs to be determined to better understand if the in vitro rate of MAT2A is sufficient to maintain the metabolic supply of SAM in different contexts . Third, the mechanisms by which cellular SAM concentrations are regulated beyond the METTL16-mediated regulation of MAT2A expression require further investigation . Additionally, the functional significance of MAT2A's nuclear localization and its reported role as a transcriptional regulator separate from its enzymatic function warrants deeper exploration . Finally, the structural dynamics of MAT2A during catalysis, particularly the conformational changes that might occur upon substrate binding and product release, would provide valuable insights into the enzyme's mechanism.

What technological advances could enhance MAT2A research?

Several technological advances could significantly enhance MAT2A research. Time-resolved structural studies, such as time-resolved X-ray crystallography or cryo-electron microscopy, could provide insights into the conformational changes of MAT2A during its catalytic cycle, helping to better understand the ordered binding mechanism and product release . Advanced nuclear magnetic resonance (NMR) techniques could be employed to study the dynamics of MAT2A-substrate interactions and allosteric regulation by MAT2B or inhibitors . Quantitative proteomics approaches would be valuable for determining the absolute concentrations of MAT2A and MAT2B in different cell types and under various physiological conditions, addressing the question of whether in vitro kinetic parameters are relevant to cellular contexts . CRISPR-based genetic screens could identify additional regulators of MAT2A function beyond METTL16 . Finally, development of more sensitive and high-throughput assays for measuring MAT2A activity and SAM production in cellular contexts would facilitate both basic research and drug discovery efforts targeting this enzyme.

How might a deeper understanding of MAT2A contribute to precision medicine approaches?

A deeper understanding of MAT2A could significantly contribute to precision medicine approaches, particularly in cancer treatment. Since MAT2A is expressed in cancer cells and has been validated as an anti-cancer target, characterizing patient-specific variations in MAT2A expression, activity, or regulation could help identify individuals most likely to respond to MAT2A inhibitors like AG-270 . Additionally, understanding how MAT2A activity and SAM production relate to specific cancer types or subtypes might allow for more targeted therapeutic strategies . The observation that MAT2B binds to the same allosteric site as known MAT2A inhibitors suggests that measuring MAT2B expression in patient samples might predict response to allosteric inhibitors, as patients with high MAT2B expression might exhibit reduced efficacy due to competition . Furthermore, understanding the METTL16-mediated regulation of MAT2A expression in response to SAM levels might reveal additional therapeutic targets or biomarkers for predicting treatment response . Finally, since MAT2A produces SAM, which is involved in numerous methylation reactions, linking MAT2A activity to specific methylation patterns in cancer could potentially identify epigenetic biomarkers for patient stratification or monitor treatment response.