Spinach enzyme

D17699C, Spinach Enzymes Neutrahze Explosives, 1998 D177000, Information Sheet... 

Wilson, E. (1998) Spinach enzymes break down explosives. Chem. Eng. News, 76, 40. 

A similar alkaline FDPase has also been obtained in highly purified form from Euglena gracilis (99) by heating and fractionation on DEAE-cellulose. The specific activity of the best preparation was approximately one-half that reported by Racker and Schroeder for the spinach enzyme. The enzyme appeared to be specific for fructose diphosphate, although SDP and ribulose diphosphate were not tested. The enzyme also required Mg2+ and was most active at pH 8.3 it showed very little activity at pH 7.5 or below. 

Boger m) showed that the transhydrogenase activity catalyzed by ferredoxin-NADP reductase obtained from Bumilleriopsis filiformis, which is very similar to the spinach enzyme, is regulated by ferredoxin and that one common nicotinamide nucleotide binding site is involved in both the diaphorase and the transhydrogenase reactions. 

Figure 4 (left page). Alignment of amino acid sequences around Lys-21 and Lys-305 and the Sr values of RuBisCO. Numbering of the residues is for the spinach enzyme. 

The Sr value of the Galdieria enzyme was 238 2.5-fold that of the spinach enzyme [18]. The value increased to 360 at 15 C. Cyanidium also had the similar enzyme. The Cm for CO2 of RuBisCO from these red algae was 6.6 to 6.7 pM the affinity of this enzyme was 2 times higher than that of the spinach enzyme. This is the surprising results because it has been believed that the plant enzyme should be most evolved in the nature. 

For the spinach enzyme, the carboxylation shows a relatively large carbon isotope effect of 1.029 with normal substrate and 1.021 with deuterated substrate (86). The isotope effect is smaller for the R. rubrum enzyme (87). Isotope effects are independent of RuBP concentration and nearly independent of pH (86). This indicates that hydrogen transfer and carboxylation are separate steps in the enzymic mechanism (88). 

The properties of purified sulfite reductase from spinach have been investigated in detail using methyl viologen as an artificial electron donor (Asada, 1967 Asada e/ al., 1969). In summary the enzyme can be resolved into two fractions both of which are essential for activity. One fraction can be replaced by bovine serum albumin while the other (sulfite reductase) exhibits the characteristics of a hemoprotein, containing 0.76 g atoms of Fe per molecular weight of 84,000. Unlike the sulfite reductase from other sources (see review by Roy and Trudinger, 1970), the purified spinach enzyme contains negligible amounts of flavin and catalyzes the reduction of hy-droxylamine, but not nitrite. The sulfite reductase from Allium exhibits similar... 

Many plant tissues contain hydroxypyruvate reductase (o-glycerate dehydrogenase) but it is especially active in leaves (Stafford et al., 1954 Stafford and Magaldi, 1954). Tolbert et al. (1970) purified the spinach enzyme and investigated its properties. It catalyzes reduction of hydroxypyruvate to D-glyceric acid by NADH [Eq. (3)]. Although the equilibrium is toward glyceric acid rather than hydroxypyruvate, the presence of sufficient amino donor and an appropriate transaminase would allow serine synthesis by the nonphosphorylated pathway with Eq. (3) as an intermediate step. 

The /t of the spinach enzyme for cystathionine is 250 fiM. Cystathionine is present in plant tissues at a concentration of 0.1-1 fx,M (Datko et al., 1974a). Unless this compound is highly compartmented, cystathionine-j8-lyase operates well below saturation with its substrate. 

Arginine 292 was of special interest because its counterpart in the spinach enzyme (arg295) is 3 residues removed tom the completely conserved his29i which is known to in the active site domain. 1 Accordingly, the codon for arg292 was selected for studies of mutagenesis and lys or leu, were introduced by directed mutagenesis at position 292 in die L subunit of A. nidulans RuBisCO (Fig. 2). 

FIGURE 3 Sequence conservation around serine-379. The dots indicate sequence identity with the spinach enzyme. Sp spinach. Mz maize. Tb tobacco. Cr Chlamydomonas reinhardtii. An Anacystis nidulans. Cr Chromatium vinosum, Ae Alcaligenes eutrophus. Rr, Rhodospirillwn rubrwn, (7). 

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). 

Cleavage of LF-1 D1 In the experiment shown in Fig. 3, the purified spinach enzyme was incubated with PS 11 enriched membranes prepared from LF-1 mutant of Scenedesmus. The D1 protein of LF-1 mutant was cleaved to the size of mature protein in the wild type, sugesting that the processing enzyme is common between higher plant and algae. 

The base in the nucleotide may be either guanine or hypoxanthine in the reaction with the heart muscle enzyme, while the spinach enzyme is specific for adenine. In crude heart muscle preparations nucleoside diphosphate kinase permits catalytic amounts of GTP or ITP to support a-ketoglutarate oxidation in the presence of ADP, which is converted to ATP. 

The central third seems most likely to be responsible for the fatty acyl specificities in the native enzymes. Indeed, the XPX GPATs all have greater preferences for 18 1 than any XQX enzyme (Fig.l). By dividing the central third approximately halfway, the chimera P2P was created whose specificity was very similar to that of the wild-type spinach enzymes. Although, most substitutions had some affect, the amino half of the central third, between residues 126 and 197 of the 370 amino-acid protein, appears to contain the structural feature(s) which dictates GPAT s fatty acid specificity. 

Rhizobitoxine, an analogue of cystathionine produced by certain strains of Rhizobium japonicum (Owens et al., 1972), is a potent irreversible inhibitor of plant cystathionine 8-lyase both in vivo (Giovanelli et al., 1973) and in vitro. Inhibition of the purified spinach enzyme was of the active-site-directed irreversible type (Giovanelli et al., 1971), and probably involves covalent linkage of a cleavage product of rhizobitoxine to the pyridoxal phosphate prosthetic group of the enzyme. 

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. 

Fig. lA. Holo-ACP formation by spinach holo-ACP synthase as a function of time. Spinach enzyme preparation (ca. 200 Vg protein)... 

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