Isocitrate Lyase from Flax

Abstract

(3), and a variety of nematodes (2,10,22). In spite of its central importance in seedling metabolism and development, little is known about catalysis by plant isocitrate lyase.The first isolation of isocitrate lyase from plants was described by Khan et al. (23). Several molecular properties of the enzyme from flax were characterized but data were insufficient to deduce the kinetic mechanism. Moreover, nothing was reported about the structure of the active site. Information about these characteristics of the plant enzyme has remained sketchy. Such information assumes special significance because plant isocitrate lyase functions within cytoplasmic organelles called glyoxysomes (3) whereas the prokaryotic enzyme is operationally 'soluble' (27, 28 (36) with a slight modification. In a total volume of 1.0 ml, the incubation mixture (pH 7.5) contained: 100 ,umol Mops, 5 tmol MgCl2, 2 pmol DTT, 10 ,imol trisodium DL-iSocitrate, and enzyme. After 10 min incubation at 30°C, the reaction was quenched by addition of 0.1 ml of 1 M oxalic acid. A suitable aliquot was diluted to 2.6 ml with distilled H20 followed by the addition of 0.1 ml of 1% (w/v) phenylhydrazine-hydrochloride. After thorough mixing, the tubes were chilled in ice for 10 min, and 1.5 ml prechilled concentrated HCI added with rapid mixing followed after 5 min by addition of 0.1 ml of 5% (w/v) potassium ferricyanide. After thorough mixing and incubation for 15 min at 25°C, the color intensity was read at 520 nm. Reaction rates were proportional to enzyme and the rate was constant over a 25-min incubation period.To examine the inhibition of isocitrate lyase by glyoxylate, the reaction rate was determined by assaying succinate using succinic dehydrogenase (38).The condensation reaction catalyzed by isocitrate lyase was studied as decribed by Johanson et al. (19) by coupling with an excess of NADP+-isocitrate dehydrogenase. Amino Acid Analysis. Isocitrate lyase was exhaustively dialyzed against 0.1 M NaCl prior to amino acid analysis. Aliquots of the dialyzed enzyme were transferred to hydrolysis vials having norleucine as an internal standard and the contents lyophilized. After addition of 0.5 ml of 6 N HCI, the vials were degassed in vacuo and sealed under N2. The hydrolysis was carried out at 1 10°C and all hydrolysates were analyzed with a Beckman 120C automatic amino acid analyzer by the method of Moore and Stein

Purification and regulatory properties of isocitrate lyase from Escherichia coli ML308

“…are inconsistent with the ordered mechanism (glyoxylate binding first) that has been proposed for other isocitrate lyases [21,29,30]. The ordered mechanism predicts that phosphoenolpyruvate should be an uncompetitive inhibitor with respect to glyoxylate.…”

“…creased with pH in the neutral pH range, being 63 + 4 /tM hanism of E. coli isocitrate lyase is thus (n = 4) at pH 7.3, 32 + 5 /tM (n = 3) at pH 6.8 and 7 ,tM ordered mechanism reported for this (single determination) at pH 6.3. As has been shown for udomonas indigofera and Neurospora isocitrate lyase from other sources [33,34], the interthe enzyme from Linum usitatissimum actions of other effectors with isocitrate lyase were also inhibition patterns given by succinate, markedly dependent on pH; for example, the K, for hosphoenolpyruvate were similar to phosphoenolpyruvate in the cleavage reaction was , even though the authors favoured 0.91 mm at pH 7.3, but only 0.1 mm at pH 6.8, and the der mechanism [30]. Phosphoenol-corresponding values for succinate were 1.12 mm and Libitor of all the isocitrate lyases that 0.3 mM.…”

“…The kinetic mechanism was probed further by using dead-end inhibitors. Phosphoenolpyruvate has been used as an analogue of succinate in studies of other isocitrate lyases [21,29,30]; it inhibited the E. coli enzyme competitively with respect to succinate and non-competitively [31] with respect to glyoxylate (Fig. 3).…”

Identification of a cysteine residue at the active site of Escherichia coli isocitrate lyase

“…2 (-). Km values for DS-isocitrate were obtained in this study (d) (flax), it was part of the glyoxylate subsite [18,21]. Nevertheless, the results suggested that bromopyruvate acts as an affinity labelling reagent for isocitrate lyase.…”

Site-directed mutagenesis of cysteine-195 in isocitrate lyase from Escherichia coli ML308

“…Several studies have identified possible active-site residues in ICL which may be important in catalysis and/or substrate binding. Inhibition of ICL from several organisms with diethyl pyrocarbonate (DEPC) [13][14][15] or Rose Bengal [16,17] implicated an active-site histidine residue. Ko et al [18] identified two histidine residues in the E. coli enzyme which reacted with DEPC, namely histidine-266 and histidine-306.…”