Cooperativity in the mechanism of malate dehydrogenase

Zimmerle, Chris T.; Alter, Gerald M.

doi:10.1021/bi00210a025

Cited by 9 publications

(5 citation statements)

References 30 publications

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“…Second, when a variety of amines were tested as catalysts for OAA decarboxylation, saturation curves were obtained despite the fact that the lowest concentration of malate used was 1.0 mM. Since the K M value of malate for malate dehydrogenase is 1.0 mM, only the upper portion of the saturation curve would have been observed at the malate concentrations used. Third, when the reaction was run with three different concentrations of malate dehydrogenase with malate held at a saturating concentration of 200 mM the rate did not change, indicating that the saturation phenomena were involved with the amine catalysis of OAA.…”

Section: Resultsmentioning

confidence: 99%

Kinetic Mechanism and Structural Requirements of the Amine-Catalyzed Decarboxylation of Oxaloacetic Acid

2008

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The kinetic and chemical mechanism of amine-catalyzed decarboxylation of oxaloacetic acid at pH 8.0 has been reevaluated using a new and versatile assay. Amine-catalyzed decarboxylation of oxaloacetic acid proceeds via the formation of an imine intermediate, followed by decarboxylation of the intermediate and hydrolysis to yield pyruvate. The decrease in oxaloacetic acid was coupled to NADH formation by malate dehydrogenase, which allowed the rates of both initial carbinolamine formation (as part of the imination step) and decarboxylation to be determined. By comparing the rates observed for a variety of amines and, in particular, diamines, the structural and electronic requirements for diamine-catalyzed decarboxylation at pH 8.0 were identified. At pH 8.0, monoamines were found to be very poor catalysts, whereas some diamines, most notably ethylenediamine, were excellent catalysts. The results indicate that the second amino group of diamines enhances the rate of imine formation by acting as a proton shuttle during the carbinolamine formation step, which enables diamines to overcome high levels of solvation that would otherwise inhibit carbinolamine, and thus imine, formation. The presence of the second amino group may also enhance the rate of the carbinolamine dehydration step. In contrast to the findings of previous reports, the second amino group participates in the reaction by enhancing the rate of decarboxylation via hydrogen-bonding to the imine nitrogen to either stabilize the negative charge that develops on the imine during decarboxylation or preferentially stabilize the reactive imine over the unreactive enamine tautomer. These results provide insight into the precise catalytic mechanism of several enzymes whose reactions are known to proceed via an imine intermediate.

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Section: Resultsmentioning

confidence: 99%

Kinetic Mechanism and Structural Requirements of the Amine-Catalyzed Decarboxylation of Oxaloacetic Acid

2008

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“…However, proving the existence of an active monomer has been controversial. Similarly, studies of cMDH have pointed toward a form of cooperativity in the catalytic mechanism that again implies some interaction of the active-site apparatus through the dimer interface (Zimmerle & Alter, 1993).…”

Section: Discussionmentioning

confidence: 99%

Engineering the quaternary structure of an enzyme: Construction and analysis of a monomeric form of malate dehydrogenase from Escherichia coli

1994

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The citric acid cycle enzyme, malate dehydrogenase (MDH), is a dimer of identical subunits. In the crystal structures of 2 prokaryotic and 2 eukaryotic forms, the subunit interface is conformationally homologous. To determine whether or not the quaternary structure of MDH is linked to the catalytic activity, mutant forms of the enzyme from Escherichia coli have been constructed. Utilizing the high-resolution structure of E. coli MDH, the dimer interface was analyzed critically for side chains that were spatially constricted and needed for electrostatic interactions. Two such residues were found, D45 and S226. At their nearest point in the homodimer, they are in different subunits, hydrogen bond across the interface, and do not interact with any catalytic residues. Each residue was mutated to a tyrosine, which should disrupt the interface because of its large size. All mutants were cloned and purified to homogeneity from an mdh-E. coli strain (BHBI 11). Gel filtration of the mutants show that D45Y and D45Y/S226Y are both monomers, whereas the S226Y mutant remains a dimer. The monomeric D45Y and D45YIS226Y mutants have 14,000-and 17,500-fold less specific activity, respectively, than the native enzyme. The dimeric S226Y has only 1.4-fold less specific activity. All forms crystallized, indicating they were not random coils. Data have been collected to 2.8 A resolution for the D45Y mutant. The mutant is not isomorphous with the native protein and work is underway to solve the structure by molecular replacement.Keywords: dimer interface; malate dehydrogenase; mdh-E. coli strain; monomeric MDH; site-directed mutagenesis; subunit dissociation Malate dehydrogenase is an enzyme that plays an important role in the citric acid cycle. The MDHs utilize the cofactor NAD to catalyze the reversible oxidation of malate to oxaloacetate. In the direction of oxaloacetate production, the reactants are NAD and malate, the dicarboxylic acid, and the products are NADH, oxaloacetate, and a proton. All known MDHs are active as dimers of identical subunits. In oligomeric enzymes, the role the Reprint requests to: Leonard J. Banaszak, Department of Biochemistry, 4-225 Millard,

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“…This process could increase the cytoplasmic NADWNAD ratio, resulting in an important contribution to drive lactate production via glycolysis for NAD restoration (Lanks, 1986;Petch and Butler, 1994). This theory could be applied presuming that an active malate shunt exists and an electron transfer system-like the NAD+linked MDH-is present in the cytosol, as described in the literature (Lanks, 1986;Mancuso et al, 1994;Sharfstein et al, 1994;Zimmerle and Alter, 1993). At the same time, the glycerol-phosphate or malate/aspartate shuttles have to show very low or no activity.…”

Section: This Significant Difference Between Insect Cells Andmentioning

confidence: 99%

Comparative analysis of glucose and glutamine metabolism in transformed mammalian cell lines, insect and primary liver cells

Neermann

Wagner

1996

J. Cell. Physiol.

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Glucose and glutamine metabolism in several cultured mammalian cell lines (BHK, CHO, and hybridoma cell lines) were investigated by correlating specific utilization and formation rates with specific maximum activities of regulatory enzymes involved in glycolysis and glutaminolysis. Results were compared with data from two insect cell lines and primary liver cells. Flux distribution was measured in a representative mammalian (BHK) and an insect (Spodoptera frugiperda) cell line using radioactive substrates. A high degree of similarity in many aspects of glucose and glutamine metabolism was observed among the cultured mammalian cell lines examined. Specific glucose utilization rates were always close to specific hexokinase activities, indicating that formation of glucose-6-phosphate from glucose (catalyzed by hexokinase) is the rate limiting step of glycolysis. No activity of the key enzymes connecting glycolysis with the tricarboxylic acid cycle, such as pyruvate dehydrogenase, pyruvate carboxylase, and phosphoenolpyruvate carboxykinase, could be detected. Flux distribution in BHK cells showed glycolytic rates very similar to lactate formation rates. No glucose- or pyruvate-derived carbon entered the tricarboxylic acid cycle, indicating that glucose is mainly metabolized via glycolysis and lactate formation. About 8% of utilized glucose was metabolized via the pentose phosphate shunt, while 20 to 30% of utilized glucose followed pathways other than glycolysis, the tricarboxylic acid cycle, or the pentose phosphate shunt. About 18% of utilized glutamine was oxidized, consistent with the notion that glutamine is the major energy source for mammalian cell lines. Mammalian cells cultured in serum-free low-protein medium showed higher utilization rates, flux rates, and enzyme activities than the same cells cultured in serum-supplemented medium. Insect cells oxidized glucose and pyruvate in addition to glutamine. Furthermore, insect cells produced little or no lactate and were able to channel glycolytic intermediates into the tricarboxylic acid cycle. Metabolic profiles of the type presented here for a variety of cell lines may eventually enable one to interfere with the metabolic patterns of cells relevant to biotechnology, with the hope of improving growth rate and/or productivity.

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Cooperativity in the mechanism of malate dehydrogenase

Cited by 9 publications

References 30 publications

Kinetic Mechanism and Structural Requirements of the Amine-Catalyzed Decarboxylation of Oxaloacetic Acid

Kinetic Mechanism and Structural Requirements of the Amine-Catalyzed Decarboxylation of Oxaloacetic Acid

Engineering the quaternary structure of an enzyme: Construction and analysis of a monomeric form of malate dehydrogenase from Escherichia coli

Comparative analysis of glucose and glutamine metabolism in transformed mammalian cell lines, insect and primary liver cells

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