A stereospecific 2-keto-4-hydroxyglutarate aldolase from Escherichia coli

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/32931/1/0000313.pd

Identity and some properties of the -threonine aldolase activity manifested by pure 2-amino-3-ketobutyrate ligase of Escherichia coli

2-Amino-3-ketobutyrate ligase catalyzes the reversible, pyridoxal 5'-phosphate-dependent condensation of glycine with acetyl CoA forming the unstable intermediate, 2-amino-3-ketobutyrate. Several independent lines of evidence indicate that the pure protein obtained in the purification of this ligase from Escherichia coli also has -threonine aldolase activity. The evidence includes: (a), a constant ratio of specific activities (aldolase/ligase) at all stages of purifying 2-amino-3-ketobutyrate ligase to homogeneity; (b), the same rate of loss of aldolase and ligase activities during controlled heat inactivation of the pure protein at 60[deg]C in the absence, as well as in the presence of acetyl CoA, a protective substrate; (c), ratios of the two enzymic activities that are not significantly different during slow inactivation by iodoacetamide, with and without -threonine added; (d), coincident rates of loss and essentially identical rates of recovery of aldolase activity and ligase activity during resolution of the holoenzyme with hydroxylamine followed by reconstitution with pyridoxal 5'-phosphate. No aldolase activity is observed with -threonine as substrate and -allothreonine is about 25% as effective as -threonine. Whereas ligase activity has a sharp pH optimum at 7.5, the aldolase activity of this pure protein is maximal at pH 9.0. Comparative apparent Km values for glycine (ligase) and -threonine (aldolase) are 10 mM and 0.9 mM, respectively, whereas corresponding respective Vmax values were found to be 2.5 [mu]mol of CoA released/min per mg vs. 0.014 [mu]mol of acetaldehyde formed (NADH oxidized)/min per mg.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/30640/1/0000282.pd

Seed germination studies I. Purification and properties of an [alpha]-amylase from the cotyledons of germinating peas

A starch-hydrolyzing enzyme present in extracts of the cotyledons of germinating peas has been purified over 3400-fold. Several independent criteria show that this activity is due to an amylase of the [alpha]-type ([alpha]-1,4-glucan-4-glucanohydrolase, EC 3.2.1.1). The enzyme exhibits maximal activity in the pH range 5.3-5.9; the Michaelis constant (Km) and energy of activation (Ea) for the enzyme-catalyzed reaction are 2[middle dot]10-4 g soluble starch per ml and 7600 cal/mole, respectively. Calcium ions protect the amylase against heat inactivation, whereas incubation of the enzyme with 5[middle dot]10-4 M EDTA for 20 min at room temperature results in complete loss of activity. With amylose and amylopectin as substrates, this enzyme is similar in action pattern to other known plant [alpha]-amylases.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/33426/1/0000828.pd

2-Amino-3-ketobutyrate CoA ligase of Escherichia coli: stoichiometry of pyridoxal phosphate binding and location of the pyridoxyllysine peptide in the primary structure of the enzyme

Pure 2-amino-3-ketobutyrate CoA ligase from Escherichia coli, which catalyzes the cleavage/condensation reaction between 2-amino-3-ketobutyrate (the presumed product of the -threonine dehydrogenase-catalyzed reaction) and glycine + acetyl-CoA, is a dimeric enzyme (Mr = 84 000) that requires pyridoxal 5'-phosphate as coenzyme for catalytic activity. Reduction of the hololigase with tritiated NaBH4 yields an inactive, radioactive enzyme adduct; acid hydrolysis of this adduct allowed for the isolation and identification of [var epsilon]-N-pyridoxyllysine. Quantitative determinations established tha t 2 mol of pyridoxal 5'-phosphate are bound per mol of dimeric enzyme. After the inactive, tritiated enzyme adduct was digested with trypsin, a single radioactive peptide containing 23 amino acids was isolated and found to have the following primary structure: Val-Asp-Ile-Ile-Thr-Gly-Thr-Leu-Gly-Ly*s-Ala-Leu-Gly-Gly-Ala-Ser-Gly-Gly-Tyr-Thr-Ala-Ala-Arg (where * = the lysine residue in azomethine linkage with pyridoxal 5'-phosphate). This peptide corresponds to residues 235-257 in the intact protein; 10 residues around the lysine residue have a high level of homology with a segment of the primary structure of 5-aminolevulinate synthase from chicken liver.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/28753/1/0000583.pd

L-threonine dehydrogenase of Escherichia coli K-12

SummaryA rapid method for purifying L-threonine dehydrogenase from Escherichia coli K-12 cells, grown on a medium containing L-threonine as carbon source, has been developed. The procedure consists of three fractionation steps of which the last is adsorption and elution of the enzyme from a column of Blue dextran-Sepharose. Homogeneous and stable samples of the dehydrogenase are obtained. L-Threonine dehydrogenase of E. coli has an average molecular weight of 140,000 and consists of 4 identical or nearly identical subunits. Km values for L-threonine and NAD[circle plus operator] are 1.4 mM and 0.19 mM, respectively; several substrate and coenzyme analogs are also active. The pH optimum is 10.3 and Mn++ stimulates dehydrogenase activity. Other properties of the pure enzyme have been established.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/22489/1/0000030.pd

Cd2+ activation of -threonine dehydrogenase from Escherichia coli K-12

Homogeneous preparations of -threonine dehydrogenase (-threonine: NAD+ oxidoreductase, EC 1.1.1.103) from Escherichia coli K-12, after having been dialyzed against buffers containing Chelex-100 resin, have a basal level of activity of 10-20 units/mg. Added Cd2+ stimulates dehydrogenase activity approx. 10-fold; this activation is concentration-dependent and is saturable with an activation Kd = 0.9 [mu]M. Full activation by Cd2+ is obtained in the absence of added thiols. The pH-activity profile of the Cd2+-activated enzyme conforms to a theoretical curve for one-proton ionization with a pKa = 7.85. Mn2+, the only other activating metal ion, competes with Cd2+ for the same binding site. Km values for -threonine and NAD+ as well as the Vmax for `demetallized', Cd2+-activated, and Mn2+-activated threonine dehydrogenase were determined and compared.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27060/1/0000050.pd

An unusual distribution of 4-substituted glutamic acids in Sophora japonica

High levels of 4-methyleneglutamine accumulate in the roots and leaves of Sophora japonica, but no detectable amounts of 4-methyleneglutamic acid and only trace quantities of 2-oxo-4-methyleneglutaric acid are seen. 4-Methylglutamic acid, however, is present in leaves and roots at a level 5-25% of that found for 4-methyleneglutamine; 2-oxo-4-methylglutaric acid is the most abundant keto acid detected in 28-day leaf extracts, but no 4-methylglutamine is seen. Transamination by pig heart glutamate: oxalacetate aminotransferase of the 2-oxo-4-methylglutaric acid that occurs in this species yields erythro-4-methylglutamic acid; the 2-oxo acid, therefore, has the (4R) configuration. The 4-methylglutamic acid isolated from this plant is also the erythro isomer and is probably of the (2S, 4R) configuration. This is the first report of the presence of 4-substituted glutamic acids in Sophora and the first instance where high levels of 4-methyleneglutamine are present in the absence of detectable levels of 4-methyleneglutamic acid.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27011/1/0000578.pd

The sulfhydryl content of -threonine dehydrogenase from Escherichia coli K-12: relation to catalytic activity and Mn2+ activation

When oxidized to cysteic acid by performic acid or converted to carboxymethylcysteine by alkylation of the reduced enzyme with iodoacetate, a total of six half-cystine residues/subunit are found in -threonine dehydrogenase (-threonine:NAD+ oxidoreductase, EC 1.1.1.103; -threonine + NAD+--> 2-amino-3-oxobutyrate + NADH) from Escherichia coli K-12. Of this total, two exist in disulfide linkage, whereas four are titratable under denaturing conditions by dithiodipyridine, 5,5'-dithiobis(2-nitrobenzoic acid), or p-mercuribenzoate. The kinetics of enzyme inactivation and of modification by the latter two reagents indicate that threonine dehydrogenase has no free thiols that selectively react with bulky compounds. While incubation of the enzyme with a large excess of iodoacetamide causes less than 10% loss of activity, the native dehydrogenase is uniquely reactive with and completely inactivated by iodoacetate. The rate of carboxymethylation by iodoacetate of one -SH group/subunit is identical with the rate of inactivation and the carboxymethylated enzyme is no longer able to bind Mn2+. NADH (0.5 mM) provides 40% protection against this inactivation; 60 to 70% protection is seen in the presence of saturating levels of NADH plus -threonine. Such results coupled with an analysis of the kinetics of inactivation caused by iodoacetate are interpreted as indicating the inhibitor first forms a reversible complex with a positively charged moiety in or near the microenvironment of a reactive -SH group in the enzyme before irreversible alkylation occurs. Specific alkylation of one -SH group/enzyme subunit apparently causes protein conformational changes that entail a loss of catalytic activity and the ability to bind Mn2+.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/28754/1/0000584.pd

Michael addition of thiols with 4-methyleneglutamic acid: Preparation of adducts, their properties and presence in peanuts

The thioethers, S-(4-amino-2,4-dicarboxybutyl)cysteamine, S-(4-amino-2,4-dicarboxybutyl)cysteine and S-(4-amino-2,4-dicarboxybutyl)glutathione, were synthesized by a Michael addition between 4-methyleneglutamic acid and the respective thiol. In dilute aqueous solution, the reactions exhibit second order kinetics; glutathione reacts much slower than cysteine or cysteamine. The adducts were characterized chromatographically, electrophoretically, and by their infra-red and nuclear magnetic resonance spectra. None of these thioethers was detected in peanut plants (Arachis hypogaea L.), even though large amounts of 4-methyleneglutamic acid, its amide, and glutathione are synthesized during peanut germination.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/24005/1/0000254.pd

4-Methyleneglutamine synthetase: A new amide synthetase present in germinating peanuts

Enzymatic activity which catalyzes the synthesis of 4-methyleneglutamine from 4-methyleneglutamic acid + ammonia was detected in and partially purified from cotyledons of peanut seeds germinated 5 to 7 days. This activity was separated from glutamine and asparagine synthetases by ammonium sulfate precipitation and DEAE-cellulose chromatography. The enzyme is distinct from these other amide synthetases in its substrate specificity, lack of amide/hydroxylamine exchange, and use of ammonium ion as amide donor together with formation of AMP from ATP. The activity is quite labile in solution, but is retained as a precipitate in ammonium sulfate or when frozen in 12.5% glycerol at -77[deg]C. This activity might be responsible for catalyzing the rapid synthesis of 4-methyleneglutamine which occurs in germinating peanuts.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/25265/1/0000708.pd