Allosteric Regulation
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MDH is found as a dimer and in some organisms a tetramer. MDH, like other multimeric proteins, can be regulated by allostery. Allostery refers to the phenomenon where a functional change in one part of a protein, called the allosteric site, influences the activity or properties of another distant part of the protein, known as the active site. This communication between sites allows protein function regulation in response to changes in the cellular environment or signaling cues. In multimeric proteins, allostery can occur within and between subunits, leading to cooperative or concerted behavior. Cooperative allosteric regulation means that binding a ligand or effector molecule to one subunit affects the affinity or activity of other subunits. This positive or negative cooperativity can amplify or dampen the response of the protein to ligands. For example, binding an activator molecule to one subunit may increase the affinity of adjacent subunits for their ligands, leading to enhanced protein activity. Conversely, binding an inhibitor molecule to one subunit may reduce the affinity of other subunits for their ligands, resulting in decreased protein activity.
The mechanism of allosteric regulation involves conformational changes within the protein structure. Ligand binding at the allosteric site induces structural rearrangements that propagate through the protein, ultimately affecting the active sites and modulating their functional properties. These conformational changes can involve alterations in protein stability, dynamics, or interactions between subunits. In addition, they can lead to shifts in the equilibrium between different protein conformations, resulting in changes in enzymatic activity, substrate binding affinity, or protein-protein interactions.
Allosteric regulation provides several advantages. It allows for integrating multiple signals or inputs, enabling the protein to respond to various environmental or cellular conditions. It also enables fine-tuning of protein function, ensuring appropriate responses to changing physiological requirements. Additionally, allosteric regulation can contribute to signal amplification and coordination of biochemical pathways, as the activation or inhibition of one protein can impact the activity of multiple downstream components. Overall, the allosteric regulation of multimeric proteins is a sophisticated mechanism that precisely controls protein function. Allosteric regulation ensures the proper functioning and adaptation of biological systems by modulating the interactions and activities of subunits within the protein complex.
In tumor cells, the primary energy sources are glutamine and glutamate rather than glucose. Human MDH plays a significant role in glutamate metabolism in rapidly proliferating tissues, particularly in tumor cells, through its NADH and pyruvate products. As a distinct isoform, mitochondrial MDH exhibits cooperative behavior regarding the substrate malate and functions as an allosteric enzyme. It is activated by fumarate but inhibited by ATP. These allosteric properties suggest a specific role for mitochondrial MDH in the malate and glutamine oxidation pathways within tumor cell mitochondria.
The catalytic activity of malate MDHs from both human and Ascaris suum is enhanced by fumarate. The fumarate binding site in human mitochondrial MDH has been identified, located at the dimer interface approximately 30 Å away from the active site, confirming fumarate's allosteric mechanism. Structural and kinetic studies have revealed the essential role of human mitochondrial Arg67 and Arg91 amino acid residues in the enzyme's activation by fumarate. One carboxylate group of fumarate forms bidentate ion-pair interactions with the side chain of these two crucial Arg residues. Mutation of these residues leads to the desensitization of human mitochondrial MDH to fumarate, confirming their significance in fumarate binding. These residues are conserved in A. suum mitochondrial MDH and numerous other decarboxylating MDHs that are not influenced by fumarate, indicating the involvement of additional amino acid residues in the activation mechanism of human mitochondrial MDH from human and A. suum.
On the other hand, ATP acts as an inhibitor of human mitochondrial MDH. ATP binds at an allosteric site, leading to conformational changes that reduce enzyme activity. The binding of ATP inhibits the enzyme's affinity for malate, thereby reducing its oxidative decarboxylation. This allosteric inhibition helps regulate the metabolic flux through the malate oxidation pathway. The allosteric regulation of human mitochondrial MDH by fumarate and ATP provides a means to coordinate the metabolic pathways involving malate and energy status within the cell. Fumarate, as an activator, stimulates human mitochondrial MDH activity when there is a need for increased malate oxidation or energy production. In contrast, ATP, as an inhibitor, acts to suppress human mitochondrial MDH activity when energy levels are sufficient or high.
MDH activity is tightly regulated through feedback inhibition and substrate inhibition. Feedback inhibition occurs when the end product of a metabolic pathway inhibits an enzyme involved in its own synthesis. In the case of MDH, excess oxaloacetate and NADH strongly inhibit its activity. Also, high malate concentrations can inhibit the reduction of oxaloacetate in some instances.
Several studies have highlighted the inhibitory effects of high oxaloacetate concentrations on MDH from various organisms, including Archaeoglobus fulgidus, Salinibacter ruber, and Saccharopolyspora erythraea. Similarly, NADH has been found to exert inhibitory effects on MDH in M. thermoautotrophicum, A. fulgidus, and E. coli.
MDH activity is regulated by the presence of glutamate, especially in the context of malate and glutamine metabolism. In tissues where glutamate serves as a primary energy source, such as rapidly proliferating cells or tumor cells, MDH plays a vital role in the metabolic pathways involving glutamate. When MDH catalyzes the conversion of malate to NADH and pyruvate, it generates crucial intermediates that contribute to the conversion of glutamate into other metabolites. This regulatory mechanism is particularly significant in highly active glutamate metabolism observed in rapidly growing tissues. The activation of MDH by glutamate enables efficient utilization of glutamate and its conversion into essential metabolic intermediates.
Citrate exerts its regulatory effects on MDH through specific mechanisms. In the direction of NAD+ to NADH (malate to oxaloacetate) conversion, citrate acts as an activator, promoting MDH activity. Conversely, in the direction of NADH to NAD+ (oxaloacetate to malate) conversion, citrate acts as an inhibitor, dampening MDH activity. Citrate is not thought to directly bind to the catalytic site of the enzyme but evidence points to citrate binding with a secondary or regulatory site. By binding to this site, citrate influences the equilibrium between two distinct conformations of MDH. Each conformation of the enzyme has a preference for binding either NAD+ or NADH. Citrate, however, specifically binds to the conformation of MDH that also accommodates NAD+. This interplay of citrate binding to the regulatory site and its influence on the conformational dynamics of MDH ultimately impacts the enzyme's activity. Citrate's binding alters the balance between the two conformations, affecting the affinity of MDH for its cofactors and thereby modulating the enzyme's catalytic efficiency.
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Here are a few projects that would provide complementary approaches to investigate the allosteric regulation of MDH, combining structural, genetic, and biochemical methods to deepen our understanding of how this key enzyme is controlled by allosteric factors.
Structural Studies: One project could focus on elucidating the structural basis of allosteric regulation in MDH. This would involve determining the three-dimensional structure of MDH in complex with its allosteric regulators, such as citrate or other metabolites. Techniques such as X-ray crystallography or cryo-electron microscopy could be employed to capture the conformational changes induced by the allosteric regulators. This project would provide insights into the molecular interactions and structural dynamics underlying the allosteric regulation of MDH.
Mutagenesis and Site-Directed Mutagenesis: Another project could involve generating specific mutations in the MDH gene to disrupt or enhance allosteric regulation. Mutations could be introduced at known allosteric sites or residues implicated in the allosteric control of MDH activity. The modified MDH variants could then be expressed and purified for functional analysis. Comparing the activity and regulation of the mutant MDH enzymes with the wild-type enzyme would help elucidate the roles of specific residues in allosteric regulation.
Kinetic and Enzyme Assays: A project could be designed to study the kinetic behavior of MDH in the presence of various allosteric regulators. This would involve measuring MDH activity under different conditions, such as varying concentrations of allosteric effectors or cofactors. Enzyme assays could be performed to quantify the rate of malate oxidation or the production of NADH in the presence or absence of allosteric regulators. By systematically analyzing the kinetics of MDH with different allosteric modulators, the project could reveal the precise mechanisms and regulatory patterns of allosteric control.
The Bell lab (jbell@sandiego.edu) is actively working on several interesting ongoing projects. Allosteric regulation explores the subunit interface in the canonical dimer structure of MDH which plays a significant role in cooperative binding and in a “reciprocating subunit” mechanism for the enzyme. This research area is starting to detail interactions that trigger and mediate subunit interactions. Citrate Regulation is complex in Malate Dehydrogenases, involving both activation and inhibition depending on species and isoform. While only binding at the active site is reported, Citrate does not appear to be a competitive inhibitor. Crystal structures of inhibited and uninhibited forms show two major areas (both involving alpha helices) of the protein show significant conformational differences. What triggers these conformational changes and how they impact overall function is currently unknown
Information & Resources
Review of MDH
Study of functional and allosteric sites in protein superfamilies (Acta Naturae 2015)
Relevant Publications
Malate dehydrogenase: a model for structure, evolution, and catalysis (Protein Sci 1994)
Protein Conformational Space at the Edge of Allostery: Turning a Nonallosteric Malate Dehydrogenase into an "Allosterized" Enzyme Using Evolution-Guided Punctual Mutations (Mol Biology Evolution 2022)
Function, Kinetic Properties, Crystallization, and Regulation of Microbial Malate Dehydrogenase (J Zhejiang Univ Sci B 2016)
Regulation of Mitochondrial Malate Dehydrogenase (JBC 1982)
A fragment-based approach identifies an allosteric pocket that impacts malate dehydrogenase activity (Commun Biology 2021)
Investigating the Allosteric Behavior of Malate Dehydrogenase from Escherichia coli. (MS Thesis 1999)
Kinetic studies of the regulation of mitochondrial malate dehydrogenase by citrate (Biochem J 1992)
List of Skills/ Techniques Needed: Molecular techniques, SDM, protein expression and purification, enzyme assay, biophysical techniques including fast thermal melt, circular dichroism and SAXS.
A list of adopters: Jessica Bell, Ellis Bell, Celeste Peterson, Amy Springer, Amy Parente, Joseph Provost…. (email josephprovost@sandiego.edu to add your name!)
