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

Homogeneous preparations of L-threonine dehydrogenase (L-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 microM. 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 L-threonine and NAD+ as well as the Vmax for 'demetallized', Cd2+-activated, and Mn2+-activated threonine dehydrogenase were determined and compared.

Identity and some properties of the L-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 L-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 degrees C in the absence, as well as in the presence of acetyl CoA, a protective substrate; (c), ratios of the two enzymatic activities that are not significantly different during slow inactivation by iodoacetamide, with and without L-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 D-threonine as substrate and L-allothreonine is about 25% as effective as L-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 L-threonine (aldolase) are 10 mM and 0.9 mM, respectively, whereas corresponding respective Vmax values were found to be 2.5 mumol of CoA released/min per mg vs. 0.014 mumol of acetaldehyde formed (NADH oxidized)/min per mg.

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 L-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 epsilon-N-pyridoxyllysine. Quantitative determinations established that 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-Lys*-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.

The sulfhydryl content of L-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 L-threonine dehydrogenase (L-threonine: NAD+ oxidoreductase, EC 1.1.1.103; L-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 L-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+.

Tritium exchange reactions catalyzed by 2-oxo-4-hydroxyglutarate aldolase from Escherichia coli K-12.

Tritiated water and tritiated substrates have been used to study exchange reactions catalyzed by Escherichia coli 2-oxo-4-hydroxyglutarate aldolase (4-hydroxy-2-oxoglutarate glyoxylate-lyase, EC 4.1.3.16, 2-oxo-4-hydroxyglutarate in equilibrium pyruvate + glyoxylate). With pyruvate, the enzyme catalyzes a rapid first-order exchange of all three methyl hydrogens in the absence of added acceptor aldehyde (i.e. glyoxylate). This reaction is not rate limiting for aldol condensation or cleavage; quite different pH-activity profiles for the exchange reaction versus aldol cleavage and also comparative effects that pH changes have on Km and V values for the two processes favor this conclusion. The exchange reaction with 2-oxobutyrate, a substrate analog, is stereoselective; one methylene hydrogen is removed at a 6-fold faster rate than the other but eventually both are exchanged. No tritium exchange occurs with glyoxylate.

Functional analysis of E. coli threonine dehydrogenase by means of mutant isolation and characterization.

The oxidation of L-threonine to 2-amino-ketobutyrate, as catalyzed by L-threonine dehydrogenase, is the first step in the major pathway for threonine catabolism in both eukaryotes and prokaryotes. Threonine dehydrogenase of E. coli has considerable amino-acid sequence homology with a number of Zn(2+)-containing, medium-chain alcohol dehydrogenases. In order to further explore structure/function interrelationships of E. coli threonine dehydrogenase, 35 alleles of tdh that imparted a no-growth or slow-growth phenotype on appropriate indicator media were isolated after mutagenesis with hydroxylamine. Within this collection, 14 mutants had single amino-acid changes that were divided into 4 groups: (a) amino-acid changes associated with proposed ligands to Zn2+; (b) a substitution of one of several conserved glycine residues; (c) mutations at the substrate or coenzyme binding site; (d) alterations that resulted in a change of charge near the active site. These findings uncover previously unidentified amino-acid residues that are important for threonine dehydrogenase catalysis and also indicate that the three-dimensional structure of tetrameric E. coli threonine dehydrogenase has considerable similarity with the dimeric horse liver alcohol dehydrogenase.

Sulfhydryl groups in relation to the structure and catalytic activity of 2-oxo-4-hydroxyglutarate aldolase from bovine liver.

Bovine liver 2-oxo-4-hydroxyglutarate aldolase (suggested name: 2-oxo-4-hydroxyglutarate glyoxylate-lyase catalyzing the reaction: 2-oxo-4-hydroxyglutarate in equilibrium pyruvate + glyoxylate) contains eight to ten sulfhydryl groups as determined by titration of the enzyme with either 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2) or p-mercuribenzoate in the presence of 1% sodium dodecyl sulfate. In the absence of a denaturant, all of the cysteinyl residues react with p-mercuribenzoate whereas only four are accessible to titration with Nbs2. No differences in -SH group reactivity can be detected during titration of the aldolase with p-mercuribenzoate. In contrast, two classes of sulfhydryls can be differentiated in the disulfide exchange reaction with Nbs2 in the absence of a denaturant; one -SH group (Class I) reacts rapidly whereas three additional thiols (Class II) titrate at approx. 0.1 the rate of the Class I-SH residue. Both pyruvate and glyoxylate protect one of the three -SH residues in Class II from reaction with Nbs2. Either substrate also prevents titration of one to two thiol groups by p-mercuribenzoate and decreases the rate of reaction of aldolase -SH groups with Nbs2 in 8 M urea. These ligand-induced changes in -SH reactivity provide a sensitive indication that the enzyme exists in an altered conformational state in the presence of either of its cosubstrates. Titration of the enzyme with either Nbs2 or p-mercuribenzoate results in a progressive loss of aldolase activity which is not proportional to the number of -SH groups modified. The enzyme retains 50% of the activity of the native enzyme when Class I and Class II thiols (i.e. four -SH groups total) are modified with Nbs2; 15% residual activity is still observed following titration of all of the cysteinyl residues with p-mercuribenzoate. Pyruvate and glyoxylate provide partial protection against inactivation. It is concluded that inactivation of 2-oxo-4-hydroxyglutarate aldolase by Nbs2 or p-mercuribenzoate is a consequence of alterations in protein structure which accompany modification of -SH groups. The data argue against the direct participation of an active-site thiol group in the catalytic mechanism of 2-oxo-4-hydroxyglutarate aldolase, be that aldol cleavage and condensation or beta-decarboxylation.

Dimerization occurs during the reversible acid inactivation of 2-oxo-4-hydroxyglutarate aldolase from Escherichia coli.

Exposure of Escherichia coli 2-oxo-4-hydroxyglutarate aldolase (4-hydroxy-2-oxoglutarate glyoxylate-lyase, EC 4.1.3.16) (molecular weight = 63 000) to phosphoric acid at pH 1.6 for 10 min at 4 degrees C causes 95% or greater inactivation. No significant effect on the rate or extent of inactivation is caused by varied aldolase concentrations or the presence of exogenous proteins. Chloride ion (50-100 mM) or 10 mM 2-oxo-4-hydroxyglutarate markedly decreases both the rate and extent of inactivation; good protection is also afforded by 10 mM pyruvate, glyoxylate, glyoxal, 2-oxoglutarate or 2-oxobutyrate. Whereas native aldolase has two free and three buried sulfhydryl groups, all five are exposed in the acid-inactivated enzyme and the molecular weight of this species at pH 1.6 is 126 000. Ultraviolet absorbance difference spectra, circular dichroism spectra and ultracentrifugation studies establish that the inactivation process is characterized by an alteration of secondary and tertiary structure as well as an aggregation to a dimer of the native molecule. Reactivation of enzyme activity to 60-80% of the original level is seen within 20 min at pH 6 to 8; examination of inactivation/reactivation as a function of pH indicates that these two processes occur via kinetically distinct pathways. Native and reactivated enzymes are identical in molecular weight, sulfhydryl titer, Km and alpha-helix content.

Woodward's reagent K inactivation of Escherichia coli L-threonine dehydrogenase: increased absorbance at 340-350 nm is due to modification of cysteine and histidine residues, not aspartate or glutamate carboxyl groups.

L-Threonine dehydrogenase (TDH) from Escherichia coli is rapidly inactivated and develops a new absorbance peak at 347 nm when incubated with N-ethyl-5-phenylisoxazolium-3'-sulfonate (Woodward's reagent K, WRK). The cofactors, NAD+ or NADH (1.5 mM), provide complete protection against inactivation; L-threonine (60 mM) is approximately 50% as effective. Tryptic digestion of WRK-modified TDH followed by HPLC fractionation (pH 6.2) yields four 340-nm-absorbing peptides, two of which are absent from enzyme incubated with WRK and NAD+. Peptide I has the sequence TAICGTDVH (TDH residues 35-43), whereas peptide II is TAICGTDVHIY (residues 35-45). Peptides not protected are TMLDTMNHGGR (III, residues 248-258) and NCRGGRTHLCR (IV, residues 98-108). Absorbance spectra of these WRK-peptides were compared with WRK adducts of imidazole, 2-hydroxyethanethiolate, and acetate. Peptides III and IV have pH-dependent lambda max values (340-350 nm), consistent with histidine modification. Peptide I has pH-independent lambda max (350 nm) indicating that a thiol is modified. WRK, therefore, does not react specifically with carboxyl groups in this enzyme, but rather modifies Cys-38 in the active site of TDH; modification of His-105 and His-255 does not affect enzyme activity. These results are the first definitive proof of WRK modifying cysteine and histidine residues of a protein and show that enzyme inactivation by WRK associated with the appearance of new absorptivity at 340-350 nm does not establish modification of aspartate or glutamate residues, as has been assumed in numerous earlier reports.

Evidence for the identity and some comparative properties of alpha-ketoglutarate and 2-keto-4-hydroxyglutarate dehydrogenase activity.

Enzyme preparations of pig heart and Escherichia coli are shown to catalyze a NAD+- and CoASH-dependent oxidation of 2-keto-4-hydroxyglutarate. Several independent lines of evidence support the conclusion that this hydroxyketo acid is a substrate for the well known alpha-ketoglutarate dehydrogenase complex of the citric acid cycle. The evidence includes (a) a constant ratio of specific activity values for the two substrates through several steps of purification, (b) identical elution profiles from a calcium phosphate gel-cellulose column and a constant ratio of specific activity toward the two substrates throughout the activity peak, (c) identical inactivation curves in controlled heat denaturation studies, (d) the same pH activity curves, (e) no effect on the oxidation of either keto acid by repeated freezing and thawing of dehydrogenase preparations, and (f) the same activity pattern when the E. coli complex is distributed into several fractions by sucrose density gradient centrifugation. Additionally, the same cofactors are required for maximal activity and glyoxylate inhibits the oxidation of either substrate noncompetitively. Ferricyanide-linked oxidation of 2-keto-4-hydroxyglutarate yields malate as the product and a 1:2:1 stoichiometric relationship is obtained between the amount of hydroxyketo acid oxidized, ferricyanide reduced, and malate formed.

4-methyleneglutamine amidohydrolase from peanut leaves : preparation, catalytic properties, and immunological responses of a highly purified form of the enzyme.

4-Methyleneglutamine amidohydrolase has been extracted and purified over 1000-fold from 14-day-old peanut (Arachis hypogaea) leaves by modification of methods described previously. The purified enzyme shows two bands of activity and three to four bands of protein after electrophoresis on nondenaturing gels. Each of the active bands is readily eluted from gel slices and migrates to its original position on subsequent electrophoresis. Although they are electrophoretically distinct, the two forms of the enzyme are immunologically identical by Ouchterlony double-diffusion techniques and have similar catalytic properties. Activity toward glutamine that has a threefold lower V(max) and a four-fold higher K(m) value copurifies with MeGln aminohydrolase activity. 4-Methyleneglutamine and 4-methyleneglutamic acid inhibit the hydrolysis of glutamine while glutamine inhibits 4-methyleneglutamine hydrolysis, further indicating the identity of the activity toward both substrates. Amidohydrolase activity is stimulated up to threefold by preincubation with either ionic or non-ionic detergents (0.1%) and also by added proteins (0.5% bovine serum albumin or whole rabbit serum); it is inhibited 50% by 1 millimolar borate or the glutamine analog, albizziin (10 millimolar). Rabbit antiserum to the purified peanut enzyme cross-reacts with one or more proteins in extracts of some plants but not others; in no instance, however, was 4-methyleneglutamine amidohydrolase activity detected in other species. Overall, the results support the hypothesis that 4-methyleneglutamine supplies N, via its hydrolysis by the amidohydrolase, to the growing shoots of peanut plants, whereas glutamine hydrolysis is prevented by the prepon-derance of the preferred substrate. Some results also suggest that this amidohydrolase activity may be regulated by metabolites and/or by association with other cellular components.

The complete amino acid sequence and identification of the active-site arginine peptide of Escherichia coli 2-keto-4-hydroxyglutarate aldolase.

The complete amino acid sequence of 2-keto-4-hydroxyglutarate aldolase from Escherichia coli has been established in the following manner. After being reduced with dithiothreitol, the purified aldolase was alkylated with iodoacetamide and subsequently digested with trypsin. The resulting 19 peptide peaks observed by high performance liquid chromatography, which compared with 21 expected tryptic cleavage products, were all isolated, purified, and individually sequenced. Overlap peptides were obtained by a combination of sequencing the N-terminal region of the intact aldolase and by cleaving the intact enzyme with cyanogen bromide followed by subdigestion of the three major cyanogen bromide peptides with either Staphylococcus aureus V8 endoproteinase, endoproteinase Lys C, or trypsin after citraconylation of lysine residues. The primary structure of the molecule was determined to be as follows. (formula; see text) 2-Keto-4-hydroxyglutarate aldolase from E. coli consists of 213 amino acids with a subunit and a trimer molecular weight of 22,286 and 66,858, respectively. No microheterogeneity is observed among the three subunits. The peptide containing the active-site arginine residue (Vlahos, C. J., Ghalambor, M. A., and Dekker, E. E. (1985) J. Biol. Chem. 260, 5480-5485) was also isolated and sequenced; this arginine residue occupies position 49. The Schiff base-forming lysine residue (Vlahos, C. J., and Dekker, E. E. (1986) J. Biol. Chem. 261, 11049-11055) is located at position 133. Whereas the active-site lysine peptide of this aldolase shows 65% homology with the same peptide of 2-keto-3-deoxy-6-phosphogluconate aldolase from Pseudomonas putida, these two proteins in toto show 49% homology.