Stability, malate dehydrogenase

Bonnete, F. Madern, D. Zaccai, G., Stability against denaturation mechanisms in halophilic malate dehydrogenase adapt to solvent connditions, 7. Mol. Biol. 1994, 244, 436-447... 

The work in the biosensor industry permitted the testing and proved of stability and reproducibility of enzymes, within the conditions employed in that area. Enzymes with demonstrated stability include lactate oxidase, malate dehydrogenase, alcohol oxidase, and glutamate oxidase. 

A key structural and mechanistic feature of lactate and malate dehydrogenases is the active site loop, residues 98-110 of the lactate enzyme, which was seen in the crystal structure to close over the reagents in the ternary complex.49,50 The loop has two functions it carries Arg-109, which helps to stabilize the transition state during hydride transfer and contacts around 101-103 are the main determinants of specificity. Tryptophan residues were placed in various parts of lactate dehydrogenase to monitor conformational changes during catalysis.54,59,60 Loop closure is the slowest of the motions. 

A single polypeptide chain can in theory exist in an infinite number of different conformations. However, one specific conformation generally appears to be the most stable for any given sequence of amino acids, and this conformation is assumed by the chain as it is synthesized within the cell. Thus, the primary structure of the polypeptide chain also determines its three-dimensional secondary and tertiary structures. It is conceivable that in some cases there may be several alternative conformations ("conforraers ) of a single chain that are of nearly equal stabilities and therefore these alternative forms may coexist. This possibility was first suggested to account for the heterogeneity noted in preparations of the cytoplasmic and mitochondrial isoenzymes of malate dehydrogenase and has also been proposed as an explanation of the multiple electrophoretic zones of erythrocyte acid phosphatase. However, no multiple enzyme forms have been shown unequivocally to be due to conformational isomerism. 

Further confusion in the order of cations in the Hofmeister series concerning the stability of a biomolecule (the enzyme halophilic malate dehydrogenase) arises from the reversal of the order when the cations are examined at low (< 1M) or at high concentrations (Ebel et al. 1999). The order of efficiency of cations to maintain the folded form of the protein at low concentrations is Ca + Mg + > Li ... 

Mitochondrial malate dehydrogenase (MDH) from several species has been shown to exist in several enzymically active forms which also appear to be conformational isoenzymes (Kitto et al, 1966, 1970). Kitto et al (1970) showed, in contrast to Epstein and Schechter (1968), that these MDH s were interconvertible in vitro, had the same amino acid compositions and molecular weights, but differed, once reversibly denatured, considerably in their heat stability. Similar interconversion of isoenzymes has also been observed with purified preparations of horse liver alcohol dehydrogenase (Lutstorf and von Wartburg, 1969). The question arises if such conformers are of any physiological or functional significance. It is possible that beeause of their differences in surface charge, the various conformative isoenzymes are differently bound within a cell. 

PEP carboxylase induction shows a 7-day lag period, then an increase up to about 60 short days followed by a sudden decrease. Malate enzyme increases for about 20 days, then stabilizes at a level lower than PEP carboxylase. Aspartate aminotransferase increases continuously on short days, reaching an activity higher than PEP carboxylase, but lower than malate dehydrogenase. Oscillations... 

This reaction was exploited by Tsukatani and Matsumoto (2000) in a stopped-flow FIA method. An immobilized D-malate dehydrogenase enzyme reactor was employed and the reduced enzymatic cofactor NADH that was formed was monitored fluorometrically (Xex = 340 nm = 460 nm). Due to the slow reaction rate, the flow was stopped with the sample in the reactor to increase reaction time. The intrinsic sample fluorescence was also assessed using a parallel blank reactor without immobilized enzyme. The method was validated through the analysis of red and white wine samples. The enzyme reactor stability was also evaluated and it was found that the sensitivity (evaluated as amplitude of response at a constant concentration of the analyte) gradually decreased to 60% within a week but then remained stable for a month. As D-malate cannot be present in naturally fermented wines (except for fraudulent addition), the interference of this primary substrate can be considered negligible. 

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