Peas, enzyme

D-ifereo-Pentulose (u-xylulose, tentatively identified as the osazone) was found chromatographically to be among the products of the action of a pea enzyme on glycolaldehyde and triose phosphate (prepared from D-fructose 1,6-diphosphate)similarly, 6-deoxy-D-fructose and 6-deoxy-L-sorbosc resulted from DL-lactaldehyde, sedoheptulose from n-erythrose, " 5-deoxy-D-triose phosphate, and D-fdo-heptulosan from n-xylose. ... 

The final reaction in the biosynthesis of threonine involves a /8-y rearrangement and the loss of phosphate from O-phosphohomoserine (Fig. 2). Threonine synthases have been isolated from Lemna (Schnyder et al., 1975) radish, sugarbeet (Madison and Thompson, 1975), peas (Schnyder et al., 1975 Thoen et al., 1978b), and barley (Aames, 1978). None of these enzymes has been extensively characterized but a requirement for pyridoxyl-5 -phosphate was demonstrated after partial purification of the barley and pea enzymes. Unlike several other enzymes associated with threonine synthesis, the activity of threonine synthase was not stimulated by monovalent cations. However, all of the plant enzymes are strongly activated by 5-adeno-sylmethionine (Section III,B,5). 

In most plants 0-phosphohomoserine rather than homoserine serves as the metabolic origin of the methionine branch of the aspartate pathway and as the direct precursor of threonine (Fig. 1). The synthesis of threonine from 0-phosphohomoserine is catalyzed by threonine synthase. This enzyme has been isolated from several plants and, in every case, enzyme activity was stimulated by a methionine derivative, 5-adenosylmethionine (Table VI). Enzyme activation is not as common as enzyme inhibition in regulating biosynthetic pathways. Nevertheless, the extent of activation of the plant methionine synthases can exceed 20-fold under some assay conditions. With the barley enzyme, only 60 ixM 5-adenosylmethionine was required for half-maximal activation (Aames, 1978). These characteristics are clearly indicative of a functional regulatory enzyme. Activation was considered to result from an increase in V iax rather than an increase in the affinity for the substrate, O-phosphohomoserine (Madison and Thompson, 1976). However, with the pea enzyme both V ,ax and are altered in the presence of the activator (Thoen et al., 1978a). The demonstration that both pea threonine... 

Cysteine inhibited sugar beet and radish threonine synthases. It was, therefore, proposed that O-phosphohomoserine would be diverted toward methionine synthesis as cysteine inhibited the enzyme (Madison and Thompson, 1976). Effective regulation could be achieved by the opposing effects of the methionine precursor, cysteine, and the methionine derivative, 5-adenosylmethionine. However, this can not be considered a universal regulatory pattern for plant threonine synthases since the barley enzyme was not inhibited by cysteine (Aarnes, 1978) and the effects of cysteine on the activity of the pea enzyme were questionable (Thoen et al., 1978b). 

Consistent with this circumstantial evidence is the effect of histidine in inhibiting isolated ATP-phosphoribosyltransferase (Wiateret al., 1971a). The enzyme from fresh extracts of pea and oat shoots was inhibited by about 50% in the presence of 0.01 mM histidine, however, when the pea enzyme was stored at 4°C overnight the sensitivity of the razyme increased to 80% although the activity in the absence of histidine dropped. The results could be explained by the inactivation overnight of an insensitive form of the enzyme and more studies are required on the control mechanisms of this enzyme. These preUminary studies have however shown that the plant enzyme is of greater sensitivity than the enzyme from Saccharomyces cerevisiae (inhibited 50% by 0.06 mM histidine). 

MW of 33-34 kDa. This concurs with an apparent MW of 35 kDa from the elution profile during size exclusion chromatography (not shown). The purest fractions containing protease activity are contaminated by a minor component of 40 kDa, but this does not co-elute with protease activity. A gel showing purification of the protease from pea leaves is shown in Fig. 2. In this case there are several bands in the purest preparation, none of which can be readily identified as the protease. The apparent MW of the pea enzyme from size exclusion chromatography was also 30-35 kDa. The spinach enzyme has a MW of 34 kDa as judged from size exclusion chromatography (4). 

Amino acid compositions for soybean and pea enzymes have been studied by several groups (see Veldink et al., 1977). Although there is general agreement for most amino acids, various values have been reported for the cysteine and cystine contents, ranging, for the soybean enzyme, from 4 (Mitsuda et al., 1967a) to 12 (Axelrod, 1974) half-cystine residues. Amino acid analyses of two isoenzymes from pea have been obtained by Eriksson and Svensson (1970) and Arens et al. (1974). 

In storage organs of other seeds a similar pattern of events can occur. In cotyledons (and embryos) of Cicer arietinum (chick-pea), enzymes of the glycolysis and fermentative pathways rise over the first 24 h after imbibition starts and then decline [41]. Enzymes of other pathways were not measured. In radish cotyledons, however, enzymes of the oxidative pentose phosphate pathway are present in the dry seed to a high level and rise even further over several days after imbibition. In contrast, the activity of the glycolytic enzyme phosphofructokinase never rises above the low level present in dry seed [108]. 

Differences between the mitochondrial G3P-AT from potato tubers and pea leaves were not only observed with respect to their intraorganelle localization but also in regard of their fatty acid specificities. While the potato enzyme showed similar acylation rates with the different acyl-CoA thioesters, the pea enzyme exhibited distinctly higher activities with saturated than with unsaturated acyl-CoA thioesters (Fig. 1A). Thus, the mitochondrial G3P-AT from pea leaves, in contrast to that from potato tubers, possesses a pronounced fatty acid specificity for saturated acyl groups. Such a specificity has not been observed for G3P-ATs from other compartments. 

As previously reported by other workers (Frentzen, etal., 1983), we find that the spinach enzyme quite efficiently selects 18 1-ACP in preference to 16 0-ACPfrom a mixture containing any of several combinations of the two. In agreement with Frentzen, etal. (1983), we also find that the pea enzyme is less selective for the unsaturated acyl substrate than is the spinach ATI. 

Tewfik and Stumpf (347) found aldolase to be present in all plant species and tissues studied. Stumpf (338) purified pea aldolase and found no requirement for divalent cations this enzyme thus resembles that found in muscle, rather than that in yeast (381), bacteria (28,124), and molds (189). Like aldolase from muscle, the pea enzyme will catalyze the condensation of dihydroxyacetone phosphate with any one of a number of aldehydes. The condensation reactions catalyzed by pea aldolase have been studied by Hough and Jones (185 and Gorin and Jones (137). 

The enzyme has also been purified from pea epicotyls and characterized (Slaughter and Davies, 1968a,b) although the pea enzyme has not been implicated in the supply of precursors for ureide biogenesis. The reaction catalyzed by the nodule enzyme was reversible, with a pH optimum of 9.4 for the forward reaction and 6.1 for the reverse reaction. values for the forward reactions were 0.25 mM for NAD and 0.29 mM for D-3-phosphoglycerate at the pH optimum, and values for the reverse reaction at pH 7.5 were 0.012 m Af for... 

De Martinis et al. (1981) have used a transition state analog coupled to a chromatographic support to purify OCT from animal and plant sources. Using this technique homogeneous preparations of OCT were obtained from animal tissues however, preparations of the pea enzyme purified 750-fold still contained one major contaminant. The enzyme has been purified to apparent homogeneity from Neurospora (Bates et al., 1985) and Alnus (Martin et al., 1983). Eid et al (1974) separated two forms of OCT from pea seedlings. Based on the suggestion by Oaks and Bidwell (1970) that two pools of ornithine are present in plants and on the occurrence of multiple forms of OCT in Pseudomonas and Bacillus, Eid et al (1974) proposed that the isoforms in peas play... 

The enzyme PYRR-DH has been characterized in pea and oat seedlings (Flores and Filner, 1985b). The pea enzyme has a higher temperature optimum (45 °C) than oat PYRR-DH (40°C pH optimum 7.5) and a broader pH optimum range (7.5-8.0). Both enzymes are specific for pyrroline and are strictly NAD dependent. Enzyme activities are inhibited by the NAD analogs thioni-cotinamide and aminopyridine dinucleotide. These properties are similar to those reported in bacteria (Jacoby and Fredericks, 1959). In addition to pea and oat, PYRR-DH activity was also detected in com, barley, soybean, and broad-bean (Hores and Filner, 1985b). 

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