Fumarase yeast

There is still a third possible mechanism for the fumarate hydratase reaction. The proton and hydroxyl groups may be added simultaneously in a concerted reaction. However, observed kinetic isotope effects are not consistent with this mechanism. In 1997 the structure of fumarase C of E. coli was reported. Each active site of the tetrameric enzyme is formed using side chains from three different subunits. The H188 imidazole is hydrogen bonded to an active site water molecule and is backed up by the E331 carboxy-late which forms a familiar catalytic pair. However, these results have not clarified the exact mode of substrate binding nor the details of the catalytic mechanism. Structural studies of fumarate hydratase from yeast and the pig are also in progress. 

Cell fractionation by mechanical rupture has already come under investigation. Two separate studies of mechanical rupture of yeast showed different rates of release for enzymes in different cell locations (13,14). Wall-linked and periplasmic enzymes were released relatively faster than total protein, soluble cytoplasmic enzymes at about the same rate, and the mitochondrial enzyme fumarase later than total protein (13). Proteolysis by the yeast s own enzymes was not found to be a problem. Activities of the released enzymes declined slowly or not at all when disruption was continued after the end of protein release, and the effect of shear was not separated from the effect of proteolysis. Shetty and Kinsella (15) also found a low rate of proteolysis after mechanical disruption, though thiol reagents added to weaken the cell walls before disruption caused an important increase in the extent of protein breakdown. 

Cell Fractionation Simulation. The wall protein, cytosol and organelles of yeast each contain enzymes which are found nowhere else in the cell. Some examples of these enzymes include invertase in the walls, glycolytic pathway enzymes in the cytosol and fumarase in the mitochondria (13). A model of recovery of these enzymes is offered here. 

Sass E, Blachinsky E, Kamiely S, Pines O. (2001). Mitochondrial and cytosolic isoforms of yeast fumarase are derivatives of a single translation product and have identical amino... 

Reactions 35 and 36 have been shown to be catalyzed by different enzyme fractions and the intermediate has been isolated and partially purified. Earlier reports had indicated that reaction 36 was a hydrolytic step resulting in the formation of arginine plus malic acid. However, recent studies indicate that the formation of malic acid was the result of the presence of fumarase in the enzyme preparation. Of considerable interest is the report that a compound similar to, if not identical with, the end product of reaction 35 is enzymatically formed from arginine plus fumaric acid by extracts of plant and animal tissues, and microorganisms. In this connection it has been reported that one of the components of the condensing enzyme system (reaction 35) is present in yeast extracts as well as in liver preparations. Although ATP is required for synthesis of the intermediate from citrulline plus aspartic acid, it is not needed for the synthesis from arginine plus fumaric acid. 

In reaction (9), succino-AICAR is cleaved to 5-amino-4-imidazolecar-boxamide ribonudeotide (AICAR) and a dicarboxyUc acid. When crude enzyme preparations were employed, a mixture of fumaric and malic acids was obtdned (1S4). Only fumaric acid was formed with a purified enzyme preparation (1S6). The presence of fumarase in the crude f ystem was undoubtedly reqmnsible for the formation of malic acid. The deaving enzyme was present in the soluble fraction of avian liver, in extracts of various microorganisms, human liver, beef liver, and bakers yeast and has been purified from chicken liver and bakers yeast. It may be identical with adenylosuccinase (1S6) (Section II, C), since the ratio of the activity of the cleaving enz]rme to adenylosuccinase activity remained constant during purification. In addition, several microbial mutants lacking adenylosuccinase also were unable to cleave succino-AICAR 1S5,1S7). 

Only a few studies in the literature reported malate production from fumarate using immobilized baker s yeast, Saccharomyces cerevisiae. Nevertheless, only one-third of the specific activity of yeast conversion is achieved as compared with the bacterial system (Oliveira et al., 1994). Neufeld et al. (1991) studied L-malate formation by immobilized S. cerevisiae that was amplified for fumarase in the presence of a surfactant. The highest specific activity... 

S. cerevisiae is studied in regard to the biochemical regulation of malic acid production (Pines et al., 1996). Under environmentally stressed conditions, a small amoxmt of fumaric acid and malic acid (less than lOg/L) was produced by this common yeast. Similar to the malic acid production pathway of Aspergillus, the cytosolic reductive pathway of acid synthesis and accumulation has been shown in S. cerevisiae. A NMR study involving glucose conversions to malic acid indicates that the following reactions lead to malic acid accumulation pyruvate oxaloacetate->fumarate malate. The involvement of cytosolic fumarase in the conversion of fumaric acid to malic acid has been corroborated. Wang et al. (1988) have shown the ability of a cytoplasmic respiratory deficient mutant of S. cerevisiae to convert fumarate to malate without the participation of mitochondrial fumarase. 

---