Identification of an essential cysteine in the reaction catalyzed by aspartate-beta-semialdehyde dehydrogenase from Escherichia coli.

The enzyme L-aspartate-beta-semialdehyde dehydrogenase from Escherichia coli has been studied by oligonucleotide-directed mutagenesis. The focus of this investigation was to examine the role of a cysteine residue that had been previously identified by chemical modification with an active site directed reagent (Biellmann et al. (1980) Eur. J. Biochem. 104, 59-64). Substitution of this cysteine at position 135 with an alanine results in complete loss of enzyme activity. However, changing this cysteine to a serine yields a mutant enzyme with a maximum velocity that is 0.3% that of the native enzyme. This C135S mutant has retained essentially the same affinity for substrates as the native enzyme, and the same overall conformation as reflected in identical behavior on gel electrophoresis and in identical fluorescence spectra. The pH profile of the native enzyme shows a loss in catalytic activity upon protonation of a group with a pKa value of 7.7. The same activity loss is observed at this pH with the serine-135 mutant, despite the differences in the pKa values for a cysteine sulfhydryl and a serine hydroxyl group that have been measured in model compounds. This observed pKa value may reflect the protonation of an auxiliary catalyst that enhances the reactivity of the active site cysteine nucleophile in the native aspartate-beta-semialdehyde dehydrogenase.

Chemical and kinetic mechanisms of aspartate-beta-semialdehyde dehydrogenase from Escherichia coli.

The chemical and kinetic mechanisms of purified aspartate-beta-semialdehyde dehydrogenase from Escherichia coli have been determined. The kinetic mechanism of the enzyme, determined from initial velocity, product and dead end inhibition studies, is a random preferred order sequential mechanism. For the reaction examined in the phosphorylating direction L-aspartate-beta-semialdehyde binds preferentially to the E-NADP-Pi complex, and there is random release of the products L-beta-aspartyl phosphate and NADPH. Substrate inhibition is displayed by both Pi and NADP. Inhibition patterns versus the other substrates suggest that Pi inhibits by binding to the phosphate subsite in the NADP binding site, and the substrate inhibition by NADP results from the formation of a dead end E-beta-aspartyl phosphate-NADP complex. The chemical mechanism of the enzyme has been examined by pH profile and chemical modification studies. The proposed mechanism involves the attack of an active site cysteine sulfhydryl on the carbonyl carbon of aspartate-beta-semialdehyde, with general acid assistance by an enzyme lysine amino group. The resulting thiohemiacetal is oxidized by NADP to a thioester, with subsequent attack by the dianion of enzyme bound phosphate. The collapse of the resulting tetrahedral intermediate leads to the acyl-phosphate product and liberation of the active site cysteine.

Crystallization and preliminary crystallographic analysis of aspartate-beta-semialdehyde dehydrogenase from Escherichia coli.

Aspartate-beta-semialdehyde dehydrogenase catalyzes the NADPH-mediated reductive dephosphorylation of beta-aspartylphosphate at a branch point in the biosynthesis of several amino acids. The enzyme from Escherichia coli has been crystallized by the vapor diffusion method from Tris buffer (pH 8.5) using polyethylene glycol 4000 as a precipitant. The crystals are orthorhombic and have the symmetry of space group P222(1), with unit cell dimensions of a = 177.8 A, b = 59.9 A, c = 118.65 A, and alpha = beta = gamma = 90 degrees. The dimensions and space group are indicative of two enzyme dimers (40 kDa per subunit) in the asymmetric unit. The crystals show strong diffraction, and a native data set has been collected to 2.5 A resolution.

Crystallization and preliminary X-ray studies of L-aspartase from Escherichia coli.

Single crystals of L-aspartate ammonia-lyase (L-aspartase) from Escherichia coli have been obtained by microdialysis at room temperature using polyethylene glycol 3350 and sodium acetate as co-precipitants. The crystals exhibit the symmetry of space group P2(1)2(1)2 with a = 156.5 A, b = 147.6 A, c = 102.5 A and diffract at least to 2.8 A.

A multinuclear NMR relaxation study of the interaction of divalent metal ions with L-aspartic acid.

Carbon-13 spin-lattice relaxation times, T1, have been measured for aqueous solutions of L-aspartic acid, L-alanine, O-phospho-L-serine, and 2-mercapto-L-succinic acid in the presence of the paramagnetic metal ions, Cu2+ and Mn2+, and Mg2+ as a diamagnetic control, at ambient temperature and neutral pH. Nitrogen-15, oxygen-17 and proton relaxation times were also obtained for L-aspartic acid and phosphorus-31 relaxation times for O-phospho-L-serine under similar conditions. The structures of these complexes in solution were determined from the various metal ion-nuclei distances calculated from the paramagnetically-induced relaxation. These results indicate that the Cu2+ interaction with L-aspartic acid is through alpha-amino and beta-carboxyl groups while Mn2+ coordinates most strongly through alpha- and beta-carboxyl groups, with the possibility of a weak interaction through the amino group. An examination of the coordination of these divalent metal ions to an analog of L-aspartic acid in which the beta-carboxyl group is replaced by a phosphate group (O-phospho-L-serine) indicated that Cu2+ coordination is now probably through the alpha-amino and phosphate groups, while this analog is a monodentate ligand for Mn2+ coordinating through the phosphate group. Removal of the beta-carboxyl group (L-alanine) also results in Cu2+ coordination through the alpha-carboxyl and alpha-amino groups, and the same ligand interactions are observed with Mn2+. Replacement of the alpha-amino group of L-aspartic acid with an -SH group (2-mercapto-L-succinate) is sufficient to eliminate any specific coordination with either Cu2+ or Mn2+.

The use of fluoro- and deoxy-substrate analogs to examine binding specificity and catalysis in the enzymes of the sorbitol pathway.

The carbohydrate specificity of the two enzymes that catalyze the metabolic interconversions in the sorbitol pathway, aldose reductase and sorbitol dehydrogenase, has been examined through the use of fluoro- and deoxy-substrate analogs. Hydrogen bonding has been shown to be the primary mode of interaction by which these enzymes specifically recognize and bind their respective polyol substrates. Aldose reductase has broad substrate specificity, and all of the fluoro- and deoxysugars that were examined are substrates for this enzyme. Unexpectedly, both 3-fluoro- and 4-fluoro-D-glucose were found to be better substrates, with significantly lower K(m) and higher Kcat/K(m) values than those of D-glucose. A more discriminating pattern of substrate specificity is observed for sorbitol dehydrogenase. Neither the 2-fluoro nor the 2-deoxy analogs of D-glucitol were found to be substrates or inhibitors, suggesting that the 2-hydroxyl group of sorbitol is a hydrogen bond donor. The 4-fluoro and 4-deoxy analogs are poorer substrates than sorbitol, also implying a binding role for this hydroxyl group. In contrast, both 6-fluoro- and 6-deoxy-D-glucitol are very good substrates for sorbitol dehydrogenase, indicating that the primary hydroxyl group at this position is not involved in substrate recognition by this enzyme.

L-aspartase: new tricks from an old enzyme.

The enzyme L-aspartate ammonia-lyase (aspartase) catalyzes the reversible deamination of the amino acid L-aspartic acid, using a carbanion mechanism to produce fumaric acid and ammonium ion. Aspartase is among the most specific enzymes known with extensive studies failing, until recently, to identify any alternative amino acid substrates that can replace L-aspartic acid. Aspartases from different organisms show high sequence homology, and this homology extends to functionally related enzymes such as the class II fumarases, the argininosuccinate and adenylosuccinate lyases. The high-resolution structure of aspartase reveals a monomer that is composed of three domains oriented in an elongated S-shape. The central domain, comprised of five-helices, provides the subunit contacts in the functionally active tetramer. The active sites are located in clefts between the subunits and structural and mutagenic studies have identified several of the active site functional groups. While the catalytic activity of this enzyme has been known for nearly 100 years, a number of recent studies have revealed some interesting and unexpected new properties of this reasonably well-characterized enzyme. The non-linear kinetics that are seen under certain conditions have been shown to be caused by the presence of a separate regulatory site. The substrate, aspartic acid, can also play the role of an activator, binding at this site along with a required divalent metal ion. Truncation of the carboxyl terminus of aspartase at specific positions leads to an enhancement of the catalytic activity of the enzyme. Truncations in this region also have been found to introduce a new, non-enzymatic biological activity into aspartase, the ability to specifically enhance the activation of plasminogen to plasmin by tissue plasminogen activator. Even after a century of investigation there are clearly a number of aspects of this multifaceted enzyme that remain to be explored.

The central enzymes of the aspartate family of amino acid biosynthesis.

The aspartate pathway is responsible for the biosynthesis of lysine, threonine, isoleucine, and methionine in most plants and microorganisms. The absence of this pathway in humans and animals makes the central enzymes potential targets for inhibition, with the aim of developing new herbicides and biocides, and also for enhancement, to improve the nutritional value of crops. Our current state of knowledge of these enzymes is reviewed, including recently determined structural information and newly constructed bifunctional fusion enzymes.

Kinetic studies of the reactions catalyzed by glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides: pH variation of kinetic parameters.

The specificity and kinetic parameters of the reactions catalyzed by glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides has been examined under a range of conditions in order to elucidate details about the mechanism of action of this enzyme. The rate of oxidation of glucose 6-phosphate is inhibited by the addition of various organic solvents. However, the low, inherent glucose dehydrogenase activity of this enzyme was stimulated under these conditions, and was further activated by divalent anions that were observed to be inhibitors of the glucose 6-phosphate dehydrogenation. From an examination of the pH variation of the enzyme kinetic parameters two groups on the enzyme that appear to be involved in the binding of the phosphate group of the sugar substrate have been detected. An enzyme catalytic group, probably a carboxylic acid, has been identified that accepts the proton from the hydroxyl group at carbon-1 of the sugar substrate during its oxidation to a lactone. The ionization of a group on the enzyme with a pK of 8.7 resulted in an increase in the maximum velocity of the glucose-6-phosphate dehydrogenase activity of the enzyme as a consequence of a pH-dependent product release step that is no longer rate limiting at high pH. Stabilization of gluconic acid-delta-lactone against nonenzymatic hydrolysis by organic solvents has allowed the kinetic parameters of the reverse reaction to be reliably measured for the first time in a narrow pH range.

Use of structural comparisons to select mutagenic targets in aspartate-beta-semialdehyde dehydrogenase.

L-Aspartate-beta-semialdehyde dehydrogenase (ASA DH) from Escherichia coli has been probed by site-directed mutagenesis to identify residues that play an important function in the catalytic activity of the enzyme. Sequence homology searching among ASA DHs that have been isolated from other species and comparisons with the structures of functionally similar D-glyceraldehyde-3-phosphate dehydrogenases (GAPDH) that have been solved from several species have been utilized to select appropriate targets for mutagenesis. A highly conserved active site glutamine has been identified in the E. coli ASA DH that enhances the reactivity of the enzyme. Alteration of this residue leads to an enzyme with reduced catalytic efficiency, yet with an unchanged binding affinity for substrates and coenzyme. Replacement of an arginine residue that is conserved throughout the ASA DH and GAPDH enzyme families leads to a significant decrease in catalytic turnover and is the only mutation examined that also results in a decreased affinity for the substrates of the reaction. This residue is assigned a role in the binding of the substrate aspartate-beta-semialdehyde. Sequence alignment of ASA DH with other NADP- and NAD-dependent enzymes has resulted in the identification of a putative pyridine nucleotide binding region. Substitution of two amino acids in this region with neutral or positively charged side chains has resulted in a change in enzyme specificity. For wild-type ASA DH, NADP is strongly favored as the coenzyme, while in this mutated enzyme the selectivity has been lowered by a factor of 60, and this enzyme has comparable affinities for either pyridine nucleotide.

Mechanism-based inactivation of L-aspartase from Escherichia coli.

The substrate analogue L-aspartate beta-semialdehyde (L-ASA) has been identified as a mechanism-based inactivator of L-aspartase from Escherichia coli. The enzyme catalyzes the deamination of L-ASA to yield fumaric acid semialdehyde (FAA) and NH4+, with the product FAA partitioning between subsequent release or irreversible enzyme inactivation. Complete protection against L-ASA inactivation is observed in the presence of the product fumarate and a divalent metal ion. However, protection against inactivation by the product FAA also requires the presence of an enzyme activator. In addition to functioning as a mechanism-based inactivator, L-ASA has also been shown to serve as an activator of L-aspartase. The mechanism of inactivation by FAA involves the attack of an active site nucleophilic at the alpha-carbon of FAA to yield a stable Michael type enzyme adduct. Subsequent formation of a hydrazone upon treatment of the enzyme adduct with 2,4-dinitrophenylhydrazine confirms the presence of the unreacted aldehydic group of FAA. Examination of a group of product analogues with different substituents has demonstrated a correlation between the electron-withdrawing ability of these functional groups and the rate of inactivation of L-aspartase.

Kinetic studies of L-aspartase from Escherichia coli: pH-dependent activity changes.

The pH dependence of the kinetic parameters of the L-aspartase-catalyzed reaction have been examined in both the amination and the deamination directions. The enzyme isolated from Escherichia coli exists in a pH-dependent equilibrium between a higher pH form that has an absolute requirement for a divalent metal ion and for substrate activation, and a low pH form that does not require activation by either substrate or metal ions. The interconversion between these enzyme forms is observed near neutral pH in the profiles examined for the reaction in either direction. This pH-dependent activation has not been observed for other bacterial aspartases. Loss of activity is observed at high pH with a pK value of 9. The pH profiles of competitive inhibitors such as 3-nitropropionic acid and succinic acid have shown that the enzyme group responsible for this activity loss must be protonated for substrate binding at the active site. An enzymatic group has also been identified that must be protonated in the amination reaction, with a pK value near 6.5, and deprotonated in the deamination reaction. This group, tentatively assigned as a histidyl residue, fulfills the criteria for the acid-base catalyst at the active site of L-aspartase.

Substrate specificity and identification of functional groups of homoserine kinase from Escherichia coli.

Homoserine kinase, an enzyme in the aspartate pathway of amino acid biosynthesis in Escherichia coli, catalyzes the conversion of L-homoserine to L-homoserine phosphate. This enzyme has been found to have broad substrate specificity, including the phosphorylation of L-homoserine analogs where the carboxyl functional group at the alpha-position has been replaced by an ester or by a hydroxymethyl group. Previous pH profile studies [Huo. X., & Viola, R. E. (1996) Arch. Biochem. Biophys. 330, 373-379] and chemical modification studies have suggested the involvement of histidinyl, lysyl, and argininyl residues in the catalytic activity of the enzyme. With the assistance of sequence alignments, several potential amino acids have been targeted for examination. Site-directed mutagenesis studies have confirmed a role for arginine-234 in the binding of the carboxyl group of L-homoserine, and the involvement of two histidine at the homoserine binding site. Mutations at these sites have led to the decoupling of the kinase activity from an inherent ATPase activity in the enzyme, and suggest the presence of independent domains for the binding of each substrate in homoserine kinase.

Specificity of aspartokinase III from Escherichia coli and an examination of important catalytic residues.

Aspartokinase III (AK III) has been purified from a plasmid-containing strain of Escherichia coli. The enzyme shows broad specificity for the phosphoryl acceptor substrate. Structural analogs of aspartic acid with a derivatized alpha-carboxyl group are accepted as alternative substrates by the enzyme. Derivatives at the alpha-amino group are also tolerated by AK III but with diminished catalytic activity. As has been previously observed with aspartokinase I (T. S. Angeles and R. E. Viola, 1992, Biochemistry 31, 799), derivatization of the beta-carboxyl group, which serves as the phosphoryl acceptor, does not prevent catalytic activity. These beta-derivatized analogs are capable of productive binding to these enzymes through a reversal of regiospecificity, making the alpha-carboxyl group available as the phosphoryl acceptor. Chemical modification and pH profile studies have identified the functional groups of cysteine and histidine as being involved in the catalytic activity of AK III.

Aldehyde-induced adenosine triphosphatase activities of fructose 6-phosphate and fructose kinases.

Chitose-6-P (2,5-anhydromannose-6-P) induces ATPase activity of fructose-6-P kinase with a Vmax 2-3% that of the normal kinase reaction with fructose-6-P or 2,5-anhydromannitol. Chitose (and presumably also chitose-6-P) is 52% hydrated in water while chitose deuterated at C-1 is 60% hydrated because of the equilibrium isotope effect of 0.73 on aldehyde hydration. Deuterated chitose-6-P gave a normal isotope effect on V/K of 1.23, but no effect on Vmax, showing that the free aldehyde is the activator and the hydrated form does not bind appreciably. With fructokinase, chitose can act either as a substrate, being phosphorylated at C-6 when adsorbed with C-6 next to MgATP, or as an inducer of ATPase activity when adsorbed with C-1 next to MgATP. The ATPase has a rate about 25% that of the kinase.

Evaluation of methods for the quantitation of cysteines in proteins.

Several methods for the quantitation of cysteines in proteins have been evaluated and compared. Titration of protein sulfhydryl groups with 5,5'-dithiobis(2-nitrobenzoate) (DTNB) under carefully controlled conditions has extended the detection limits of this method with high accuracy and reproducibility. Results are reported for a variety of enzymes containing a range of total cysteines with different degrees of solvent accessibility and reactivity. A papain amplification assay has also been examined, in which reactivation of the disulfide-blocked active site cysteine of papain can be achieved by a coupled reaction with protein sulfhydryl groups. Detection of sulfhydryls by this amplification assay can be extended, by increasing the enzyme assay times, to achieve over a 40-fold increase in sensitivity over the improved DTNB titration method. Alternatively, titration of enzyme cysteinyl residues with either bromobimane or a maleimide derivative of naphthopyranones has the advantage that a fluorescent product results upon modification of the sulfhydryl group. Reaction of bromobimane with several different enzymes results in nonspecific background fluorescence that limits the detection range of this method unless the products are separated. In contrast, low background fluorescence and high quantum yields with maleimide naphthopyranoses has allowed detection of protein cysteinyl residues with very high sensitivities.

Functional group characterization of homoserine kinase from Escherichia coli.

Homoserine kinase (EC 2.7.1.39), a key enzyme in the aspartate pathway of amino acid biosynthesis in Escherichia coli, catalyzes the phosphorylation of L-homoserine to form L-homoserine phosphate. The ThrB gene coding for this enzyme has been cloned, and the enzyme has been overexpressed and purified to homogeneity with a simplified purification scheme. An examination of the pH dependence of the V/K profile for L-homoserine shows that the enzyme loses activity upon protonation of a single functional group and upon de-protonation of a second functional group, with both groups appearing to be of the cationic acid type. Incubation of the enzyme with diethylpyrocarbonate leads to the complete loss of enzyme activity. Spectral and chemical characterization of the derivatized enzyme has shown that this activity loss is caused by the modification of a histidine residue. Treatment of the enzyme with pyridoxal-5'-phosphate also results in enzyme inactivation. The spectra evidence for the formation of a Schiff base, and the complete protection afforded by substrates and inhibitors, indicate that homoserine kinase also contains a lysine that is essential for catalytic activity.

Recovery of catalytic activity from an inactive aggregated mutant of l-aspartase.

Two highly conserved lysyl residues have been replaced with an arginine to examine their role in the mechanism of l-aspartase from Escherichia coli. Replacement of an active-site lysine results in a significant loss of catalytic efficiency [A. S. Saribas, J. F. Schindler, and R. E. Viola (1994) J. Biol. Chem. 269, 6313-6319], while replacement of the second lysine leads to a completely inactive and insoluble protein. Fluorescence spectral evidence has suggested that the loss of activity is due to the misfolding of this aspartase mutant. Some catalytic activity is recovered when the mutant is treated with varying levels of denaturants, and extended treatment with high levels of guanidine.HCl results in the recovery of a substantial fraction of the wild-type activity from this inactive mutant. However, upon removal of the denaturant this mutant enzyme slowly reverts to its inactive and insoluble form. Treatment with an artificial chaperone system in which solubilization by detergent is followed by its removal with beta-cyclodextrin leads to a stable enzyme under nondenaturing conditions with about half the catalytic activity of the wild-type enzyme. These results confirm a structural role for lysine-55 in l-aspartase and demonstrate that additional characterization is required before conclusions can be drawn from the production of an inactive mutant.

The kinetic mechanisms of the bifunctional enzyme aspartokinase-homoserine dehydrogenase I from Escherichia coli.

The kinetic mechanisms of the reactions catalyzed by the two catalytic domains of aspartokinase-homoserine dehydrogenase I from Escherichia coli have been determined. Initial velocity, product inhibition, and dead-end inhibition studies of homoserine dehydrogenase are consistent with an ordered addition of NADPH and aspartate beta-semialdehyde followed by an ordered release of homoserine and NADP+. Aspartokinase I catalyzes the phosphorylation of a number of L-aspartic acid analogues and, moreover, can utilize MgdATP as a phosphoryl donor. Because of this broad substrate specificity, alternative substrate diagnostics was used to probe the kinetic mechanism of this enzyme. The kinetic patterns showed two sets of intersecting lines that are indicative of a random mechanism. Incorporation of these results with the data obtained from initial velocity, product inhibition, and dead-end inhibition studies at pH 8.0 are consistent with a random addition of L-aspartic acid and MgATP and an ordered release of MgADP and beta-aspartyl phosphate.