Active site analysis of the potential antimicrobial target aspartate semialdehyde dehydrogenase.

Aspartate-beta-semialdehyde dehydrogenase (ASADH) lies at the first branch point in the biosynthetic pathway through which bacteria, fungi, and the higher plants synthesize amino acids, including lysine and methionine and the cell wall component diaminopimelate from aspartate. Blocks in this biosynthetic pathway, which is absent in mammals, are lethal, and inhibitors of ASADH may therefore serve as useful antibacterial, fungicidal, or herbicidal agents. We have determined the structure of ASADH from Escherichia coli by crystallography in the presence of its coenzyme and a substrate analogue that acts as a covalent inhibitor. This structure is comparable to that of the covalent intermediate that forms during the reaction catalyzed by ASADH. The key catalytic residues are confirmed as cysteine 135, which is covalently linked to the intermediate during the reaction, and histidine 274, which acts as an acid/base catalyst. The substrate and coenzyme binding residues are also identified, and these active site residues are conserved throughout all of the ASADH sequences. Comparison of the previously determined apo-enzyme structure [Hadfield et al. J. Mol. Biol. (1999) 289, 991-1002] and the complex presented here reveals a conformational change that occurs on binding of NADP that creates a binding site for the amino acid substrate. These results provide a structural explanation for the preferred order of substrate binding that is observed kinetically.

Alteration of the specificity of malate dehydrogenase by chemical modulation of an active site arginine.

Malate dehydrogenase from Escherichia coli is highly specific for the oxidation of malate to oxaloacetate. The technique of site-specific modulation has been used to alter the substrate binding site of this enzyme. Introduction of a cysteine in place of the active site binding residue arginine 153 results in a mutant enzyme with diminished catalytic activity, but with K(m) values for malate and oxaloacetate that are surprisingly unaffected. Reaction of this introduced cysteine with a series of amino acid analog reagents leads to the incorporation of a range of functional groups at the active site of malate dehydrogenase. The introduction of a positively charged group such as an amine or an amidine at this position results in improved affinity for several inhibitors over that observed with the native enzyme. However, the recovery of catalytic activity is less dramatic, with less than one third of the native activity achieved with the optimal reagents. These modified enzymes do have altered substrate specificity, with alpha-ketoglutarate and hydroxypyruvate no longer functioning as alternative substrates.

Structural analyses of a malate dehydrogenase with a variable active site.

Malate dehydrogenase specifically oxidizes malate to oxaloacetate. The specificity arises from three arginines in the active site pocket that coordinate the carboxyl groups of the substrate and stabilize the newly forming hydroxyl/keto group during catalysis. Here, the role of Arg-153 in distinguishing substrate specificity is examined by the mutant R153C. The x-ray structure of the NAD binary complex at 2.1 A reveals two sulfate ions bound in the closed form of the active site. The sulfate that occupies the substrate binding site has been translated approximately 2 A toward the opening of the active site cavity. Its new location suggests that the low catalytic turnover observed in the R153C mutant may be due to misalignment of the hydroxyl or ketone group of the substrate with the appropriate catalytic residues. In the NAD.pyruvate ternary complex, the monocarboxylic inhibitor is bound in the open conformation of the active site. The pyruvate is coordinated not by the active site arginines, but through weak hydrogen bonds to the amide backbone. Energy minimized molecular models of unnatural analogues of R153C (Wright, S. K., and Viola, R. E. (2001) J. Biol. Chem. 276, 31151-31155) reveal that the regenerated amino and amido side chains can form favorable hydrogen-bonding interactions with the substrate, although a return to native enzymatic activity is not observed. The low activity of the modified R153C enzymes suggests that precise positioning of the guanidino side chain is essential for optimal orientation of the substrate.

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.

From malate dehydrogenase to phenyllactate dehydrogenase. Incorporation of unnatural amino acids to generate an improved enzyme-catalyzed activity.

Malate dehydrogenase (MDH) from Escherichia coli is highly specific for its keto acid substrate. The placement of the active site-binding groups in MDH effectively discriminates against both the shorter and the longer keto dicarboxylic acids that could potentially serve as alternative substrates. A notable exception to this specificity is the alternative substrate phenylpyruvate. This aromatic keto acid can be reduced by MDH, albeit at a somewhat slower rate and with greatly diminished affinity, despite the presence of several substrate-binding arginyl residues and the absence of a hydrophobic pocket in the active site. The specificity of MDH for phenylpyruvate has now been enhanced, and that for the physiological substrate oxaloacetate has been diminished, through the replacement of one of the binding arginyl residues with several unnatural alkyl and aryl amino acid analogs. This approach, called site-specific modulation, incorporates systematic structural variations at a site of interest. Molecular modeling studies have suggested a structural basis for the affinity of native MDH for phenylpyruvate and a rationale for the improved catalytic activity that is observed with these new, modified phenyllactate dehydrogenases.

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.

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.

Introduction of histidine analogs leads to enhanced proton transfer in carbonic anhydrase V.

The rate-limiting step in the catalysis of the hydration of CO2 by carbonic anhydrase involves transfer of protons between zinc-bound water and solution. This proton transfer can be enhanced by proton shuttle residues within the active-site cavity of the enzyme. We have used chemical modulation to provide novel internal proton transfer groups that enhance catalysis by murine carbonic anhydrase V (mCA V). This approach involves the site-directed mutation of a targeted residue to a cysteine which is then subsequently reacted with an imidazole analog containing an appropriately positioned leaving group. Compounds examined include 4-bromoethylimidazole (4-BEI), 2-chloromethylimidazole (2-CMI), 4-chloromethylimidazole (4-CMI), and a triazole analog. Two sites in mCA V, Lys 91 and Tyr 131, located on the rim of the active-site cavity have been targeted for the introduction of these imidazole analogs. Modification of the introduced Cys 131 with 4-BEI and 4-CMI resulted in enhancements of up to threefold in catalytic activity. The pH profiles indicate the presence of a new proton shuttle residue of pKa near 5.8, consistent with the introduction of a functional proton transfer group into the active site. This is the first example of incorporation by chemical modification of an unnatural amino acid analog of histidine that can act as a proton shuttle in an enzyme.

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.

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.

Enhancement of catalytic activity by gene truncation: activation of L-aspartase from Escherichia coli.

Aspartase from Escherichia coli is activated by proteolysis at the carboxy-terminal. A systematic study has been undertaken with the goal of identifying the amino acids in this region that influence the catalytic activity of aspartase. Stop codons have been introduced at various positions to prematurely truncate the aspA gene that encodes for aspartase by sequentially eliminating each of the polar and charged amino acids in this region. The affinity of the enzyme for its substrate aspartic acid decreases systematically as each functionally significant amino acid is eliminated. However, enhanced catalytic activity (up to 2.5 times the kcat for native aspartase) is observed for those truncation mutants that end in a positively charged carboxy-terminal amino acid. The precise position of the proteolytic activation of aspartase has been defined, and this covalent activation has been shown to be independent of the allosteric activation of aspartase that is also observed.

The structure of L-aspartate ammonia-lyase from Escherichia coli.

The X-ray crystal structure of l-aspartate ammonia-lyase has been determined to 2.8 A resolution. The enzyme contains three domains, and each domain is composed almost completely of alpha helices. The central domain is composed of five long helices. In the tetramer, these five helices form a 20-helix cluster. Such clusters have also been seen in delta-crystallin and in fumarase. The active site of aspartase has been located in a region that contains side chains from three different subunits. The structure of the apoenzyme has made it possible to identify some of the residues that are involved in binding the substrate. These residues have been examined by site-directed mutagenesis, and their putative roles have been assigned [Jayasekera, M. M. K., Shi, W., Farber, G. K., & Viola, R. E. (1997) Biochemistry 36, 9145-9150].

Evaluation of functionally important amino acids in L-aspartate ammonia-lyase from Escherichia coli.

The high-resolution structure of l-aspartate ammonia-lyase from Escherichia coli has recently been determined [Shi, W., Dunbar, J., Jayasekera, M. M. K., Viola, R. E., & Farber, G. K. (1997) Biochemistry 36, 9136-9144]. An examination of the putative active site has been carried out, with the active site located in a cleft that contains the functionally significant lysine 327. A list of potential active site residues has been generated based on their proximity to this active site lysine, sequence homology comparisons with other members of the aspartase-fumarase enzyme family, and the necessity for chemically reasonable functionalities for the proposed roles. The five most likely candidates in the putative active site cleft have been examined by site-directed mutagenesis to test their feasibility for either substrate binding or acid-base catalytic roles. Arginine and lysine residues have been identified that appear to function in the orientation and binding of aspartic acid at the enzyme active site. Some tentative assignments have also been made of the acid and base catalytic groups that are proposed to be involved in the deamination reaction.

Mapping the mechanism-based modification sites in L-aspartase from Escherichia coli.

Inactivation of the enzyme L-aspartase from Escherichia coli by the substrate analog aspartate beta-semialdehyde has previously been shown to occur by the mechanism-based conversion to the corresponding product aldehyde, followed by covalent modification of cysteine-273 (F. Giorgianni et al. (1995) Biochemistry 34, 3529). Inactivation by the product analog, fumaric acid aldehyde (FAA), has now been examined directly by adding a reduction step to the modification protocol in order to stabilize the resulting enzyme-FAA derivative(s). HPLC and mass spectrometric analyses of proteolytic digests of inactivated L-aspartase have confirmed the modification at cysteine-273, and have also identified an additional modified peptide. The inactivation at this additional site involves a crosslink between cysteine-140 and an adjacent lysine. Site-directed mutagenesis studies have shown that cysteine-140 is a very reactive and accessible nucleophile that is not, however, directly involved in enzyme activity. The adjacent lysine-139 that is modified does appear to play a role in substrate binding. A double mutant in which both of the reactive cysteines have been replaced is almost completely insensitive to modification by these substrate and product analogs.

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.

Conversion of cysteinyl residues to unnatural amino acid analogs. Examination in a model system.

Improved and efficient techniques have led to an explosive growth in the application of site-directed mutagenesis to the study of enzymes. However, the limited availability of only those 20 amino acids that are translated by the genetic code has prevented the systematic variation of an amino acid's properties in order to define more precisely its role in the catalytic mechanism of an enzyme. An approach is being examined that combines the high specificity of site-directed mutagenesis with the flexibility of chemical modification to overcome these limitations. A set of reagents has been synthesized and reacted with a cysteine model to produce a series of amino acid structural analogs at appreciable rates and in good overall yields. The selective incorporation of these analogs in place of important functional amino acids in a protein will allow a more detailed examination of the role of that amino acid.

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.

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.