Bruce A. McFadden

The determination of glyoxylic acid in biological systems

Abstract A method is described for the determination of glyoxylate (or the acid) which involves potentiometric titration of the NaOH produced as a result of the formation of the sulfite addition compound. Results are in good agreement with those from a less precise assay involving periodate oxidation. Using the sulfite method to determine the concentration of glyoxylate in stock solutions, a highly specific colorimetric procedure is described for the assay of more dilute solutions. Glyoxylate has an aM of 16,300. A diversity of compounds of biological interest, including carbonyl substances, does not interfere with the determination. Of the compounds tested, only glyoxal should interfere substantially at molar concentrations equivalent to glyoxylate.

Alkylation of isocitrate lyase from Escherichia coli by 3-bromopyruvate

The inactivation of tetrameric isocitrate lyase from Escherichia coli by 3-bromopyruvate, exhibiting saturation kinetics, is accompanied by the loss of one sulfhydryl per subunit. The substrates glyoxylate and isocitrate protect against inactivation whereas the substrate succinate does not. The modification by 3-bromopyruvate (equimolar to subunits) imparts striking resistance to digestion of isocitrate lyase by trypsin, chymotrypsin, and V8 protease as well as a major decrease in the intensity of tryptophan fluorescence. After alkylation, the sequence Gly-His-Met-Gly-Gly-Lys is found following the modified Cys residue in the tryptic peptide representing positions 196-201. Thus Cys195 is alkylated by 3-bromopyruvate.

Regulation of ribulose-1,5-diphosphate carboxylase by 6-phospho-D-gluconate☆

6-phospho-D-gluconate has been found to inhibit ribulose-1,5-diphosphate (RuDP) carboxylase from several sources, including spinach, Hydrogenomonas eutropha, Chromatium strain D, Ectothiorhodospira halophila and Anacystis nidulans. Sensitivity to 6-phosphogluconate appears to correlate with molecular size, i. e., all sensitive enzymes have molecular weights larger than 500,000 daltons. Smaller RuDP carboxylases (from Rhodospirillum rubrum, Rhodopseudomonas spheroides and Thiobacillus denitrificans) are insensitive to this ligand. These and other observations suggest that 6-phosphogluconate may regulate the activity of RuDP carboxylase in the dark in green plants. Sensitivity to 6-phosphogluconate may be useful in classifying the carboxylases much as aldolases have been divided into two categories.

Isocitrate lyase from Neurospora crassa: I. Purification, kinetic mechanism, and interaction with inhibitors

Abstract After establishing conditions which stabilize isocitrate lyase (EC 4.1.3.1) in cell-free preparations of Neurospora crassa , this enzyme was purified to homogeneity. The kinetic properties of the enzyme were studied in an effort to learn about the regulation of the enzyme in vivo, the mechanism of action and the properties of the active site. The enzyme was found to be inhibited by its products succinate and glyoxylate, and by phosphoenolpyruvate, fumarate, malate and fructose 1,6-bis-phosphate. The studies on the inhibition of the forward reaction by the enzyme products, glyoxylate and succinate, and studies of the kinetics of the back reaction showed that the enzyme functioned in a uni—bi reaction mechanism with succinate leaving before glyoxylate in the cleavage reaction. The inhibitions of the enzyme by maleate and the substrate analog homoisocitrate were examined, and the results shed light on the structure of the active site. In further studies, isocitrate lyase from both N. crassa and Pseudomonas indigofera was found to be competitively inhibited by the inorganic anions SO 4 2− , HPO 4 2− , Cl − and NO 3 − . The results obtained suggest that the isocitrate lyases from N. crassa and P. indigofera may have a common evolutionary background.

Purification and properties of isocitrate lyase from flax seedlings.

Abstract Isocitrate lyase has been purified from flax ( Linum usitatissimum ) seedlings. The final preparation was homogeneous by the criteria of polyacrylamide disc gel electrophoresis, immunodiffusion, and immunoelectrophoresis. From exclusion chromatography on Sephadex G-200, the molecular weight and Stokes radius of the enzyme were 264,000 and 5.28 × 10 −7 cm, respectively. The subunit molecular weight was 67,000. Thus, the enzyme appears to be tetrameric. The enzyme required Mg 2+ and cysteine for activity. The optimal pH of the enzyme was 7.5 both in Tris and in phosphate buffers. There are three disulfide bridges and two of eight cysteine residues are buried. Inactivation of isocitrate lyase resulted from short-term modification of enzymatic thiols but this could be reversed by added thiols. The enzyme was competitively inhibited by glyoxylate, l -tartrate, and malonate in catalysis of isocitrate cleavage.

Isocitrate lyase from Pseudomonas indigofera I. Preparation, amino acid composition and molecular weight

Abstract An improved method for the purification of isocitrate lyase (Ds-isocitrate glyoxalate-lyase, EC 4.1.3.1) from Pseudomonas indigofera is described. The enzyme preparation is homogeneous by sedimentation and diffusion criteria and virtually homogeneous by gel-electrophoretic analysis. The sedimentation coefficient (s20, w), diffusion coefficient (D20, w), partial specific volume, molecular weight, extinction coefficient at 280 mμ, absorbancy ratio at 280 to 260 mμ, and turnover number of crystalline isocitrate lyase are respectively, 9.49·10−13 sec, 3.87·10−7 cm2/sec, 0.730, 2.22·105, 1.710 cm2/g, 2.03, and 7300 moles glyoxylate formed per min per mole enzyme. The amino acid composition of the enzyme was determined by ion-exchange chromatography. All of the common amino acids are present in the enzyme. The enzyme contains 21 half-cystine residues, 255 basic amino acid residues, 197 aspartic plus asparagine residues, and 240 glutamic plus glutamine residues per mole.

Isocitrate lyase from Neurospora crassa. II. Composition, quaternary structure, C-terminus, and active-site modification.

Abstract Several features of the molecular structure of Neurospora crassa isocitrate lyase ( threo - d s -isocitrate glyoxylate lyase, EC 4.1.3.1) were studied. The subunit molecular weight was found to be 67 000 by the procedure of Weber, K. and Osborn, M. (1969) J. Biol. Chem. 244, 4406–4412. The molecular weight of the native enzyme was indicated to be 270 000 by gel filtration. These values suggest that the protein is tetrameric. The enzyme was found to have a histidine in the C-terminal position and a phenylalanine in the penultimate position. The C-terminal amino acids were very important for enzyme activity. 3-Bromopyruvate was found to irreversibly inactive the enzyme with the inactivation showing saturation kinetics. Substrate or products protected against the inactivation. The amino acid content was also determined. The N. crassa isocitrate lyase was, in most characteristics, similar to the Pseudomonas indigofera isocitrate lyase. The overall similarities provide a basis for suggesting that the prokaryotic P. indigofera isocitrate lyase is homologous to the eukaryotic N. crassa isocitrate lyase.

The structure and domain organization of Escherichia coli isocitrate lyase.

Enzymes of the glyoxylate-bypass pathway are potential targets for the control of many human diseases caused by such pathogens as Mycobacteria and Leishmania. Isocitrate lyase catalyses the first committed step in this pathway and the structure of this tetrameric enzyme from Escherichia coli has been determined at 2.1 A resolution. E. coli isocitrate lyase, like the enzyme from other prokaryotes, is located in the cytoplasm, whereas in plants, protozoa, algae and fungi this enzyme is found localized in glyoxysomes. Comparison of the structure of the prokaryotic isocitrate lyase with that from the eukaryote Aspergillus nidulans reveals a different domain structure following the deletion of approximately 100 residues from the larger eukaryotic enzyme. Despite this, the active sites of the prokaryotic and eukaryotic enzymes are very closely related, including the apparent disorder of two equivalent segments of the protein that are known to be involved in a conformational change as part of the enzymes catalytic cycle.

d-ribulose-1,5-bisphosphate carboxylase in Chlorobium thiosulfatophilum Tassajara

Abstract d -Ribulose-1,5-bisphosphate carboxylase (3-phospho- d -glycerate carboxy-lyase (dimerizing), EC 4.1.1.39) was readily detected in extracts of Chlorobium thiosulfatophilium Tassajara after transatlantic air delivery. The enzyme, which was moderately unstable, had a specific activity of 7 nmoles CO 2 fixed/min per mg protein when assayed in the presence of 4 mM EDTA. It could be purified 45-fold in one step by centrifugation into a 0.2–0.8 M sucrose density gradient. The enzyme had a molecular weight of 3.61 · 10 5 when compared with standards after sedimentation into sucrose density gradients. This oligomer of the enzyme was composed of six polypeptide chains ( M r approx. 53 000) revealed by gel electrophoresis in the presence of sodium dodecylsulfate. There was no evidence for a smaller, second type of subunit observed for larger ribulose-1,5-bisphosphate carboxylases.

Particulate isocitrate lyase and malate synthase in Caenorhabditis elegans

Abstract Biochemical evidence is presented suggesting the particulate nature of some of the glyoxylate cycle enzymes in the free-living nematode Caenorhabditis elegans . A crude homogenate of freshly grown nematodes was prepared by gentle grinding. Isopycnic sucrose gradient centrifugation of the supernatant fraction obtained by low-speed centrifugation yielded four protein bands. The glyoxylate cycle enzymes, isocitrate lyase and malate synthase, appeared in the lowermost band at a density of 1.25 g/cm 3 , while the mitochondrial enzymes, fumarase and NADH oxidase, equilibrated at a density of 1.18 g/cm 3 . The glyoxylate cycle and the mitochondrial enzymes were released differentially from the particulate fraction either by sonic treatment or by treatment with 0.1% Triton X-100. The specific activities of isocitrate lyase and malate synthase in the supernatant fraction obtained after a sonic treatment of the particulate fraction were always higher than those observed in the parent fraction.