Alteration of substrate specificity of leucine dehydrogenase by site-directed mutagenesis

The residues L40, A113, V291, and V294, in leucine dehydrogenase (LeuDH), predicted to be involved in recognition of the substrate side chain, have been mutated on the basis of the molecular modeling to mimic the substrate specificities of phenylalanine (PheDH), glutamate (GluDH), and lysine dehydrogenases (LysDH). The A113G and A113G/V291L mutants, imitating the PheDH active site, displayed activities toward -phenylalanine and phenylpyruvate with 1.6 and 7.8% of k_cat values of the wild-type enzyme for the preferred substrates, -leucine and its keto-analog, respectively. Indeed, the residue A113, corresponding to G114 in PheDH, affects the volume of the side-chain binding pocket and has a critical role in discrimination of the bulkiness of the side chain. Another two sets of mutants, substituting L40 and V294 of LeuDH with the corresponding residues predicted in GluDH and LysDH, were also constructed and characterized. Emergence of GluDH and LysDH activities in L40K/V294S and L40D/V294S mutants, respectively, indicates that the two corresponding residues in the active site of amino acid dehydrogenases are important for discrimination of the hydrophobicity/polarity of the aliphatic substrate side chain. All these results demonstrate that the substrate specificities of the amino acid dehydrogenases can be altered by protein engineering. The engineered dehydrogenases are expected to be used for production and detection of natural and non-natural amino acids.     

Nickel-induced substrate inhibition of bovine liver glutamate dehydrogenase

The effects of nickel ions on reductive amination and oxidative deamination activities of bovine liver glutamate dehydrogenase (GDH) were examined kinetically by UV spectroscopy, at 27 degrees C, using 50 mM Tris, pH 7.8, containing 0.1 M NaCl. Kinetic analysis of the data obtained by varying NADH concentration indicated strong inhibition, presumably due to binding of the coenzyme to the regulatory site. In contrast, almost no inhibition was observed in the forward reaction. The fact that nickel ions have the capacity to enhance binding of NADH to the enzyme was confirmed by an electrochemical method using a modified glassy carbon electrode. Use of NADPH instead of NADH showed only a weak substrate inhibition, presumably related to lower affinity of NADPH for binding to the regulatory site. Lineweaver-Burk plots with respect to alpha-ketoglutarate and ammonium ions indicated substrate and competitive inhibition patterns in the presence of nickel ions, respectively. ADP at 0.2 mM concentration protected inhibition caused by nickel. These observations are explained in terms of formation of a nickel-NADH complex with a higher affinity for binding to the regulatory site in GDH, as compared with the situation where nickel is not present. Such effects may be important for regulation of GDH and other NADH-utilizing enzymes.     

Hyperinsulinism-hyperammonemia syndrome caused by mutant glutamate dehydrogenase accompanied by novel enzyme kinetics

Hyperinsulinism-hyperammonemia syndrome (HHS) is a recently identified genetic disorder characterized by hyperinsulinemic hypoglycemia with concomitant hyperammonemia. In patients with HHS, activating mutations in the glutamate dehydrogenase (GDH) gene have been identified. GDH is a key enzyme linking glutamate metabolism with the Krebs cycle and catalyzes the conversion of glutamate to α-ketoglutarate. The activity of GDH is controlled by allosteric inhibition by GTP and, so far, all the mutations of HHS patients have been located within the GTP-binding site. Characteristically, GDH from these individuals have therefore normal basal activity in conjunction with a loss of GTP inhibition. In this study, however, we have identified a novel variant GDH in a patient with a more severe form of HHS. The mutation is located outside the GTP-binding site and the patient’s GDH shows consistently higher activity, even in the absence of allosteric effectors. These results further support the hypothesis that the activating mutation of GDH is the cause of HHS. The mechanism leading to the activation of GDH, however, is not always related to the loss of GTP inhibition as was originally suggested. Received: 4 January 1999 / Accepted: 11 March 1999     

The regulation of glutamate dehydrogenase isoenzymes by nitrite in isolated pea roots

Unmetabolized nitrite occurs in 3 cm long apical segments of primary pea roots(Pisum sativum L., cv. Raman), isolated from 6 d old seedlings, grown under aseptic conditions for 10 h in nutrient solutions containing 0.1 to 2 mM nitrite concentration, its amount being dependent on the concentration in the nutrient solution. With increasing amount of nitrite in the roots, the ratio between NAD⁺-GDH₁ and NAD⁺-GDH₂ isoenzymes is changed without influencing significantly the total NAD⁺-GDH activity. After 48 h cultivation unmetabolized nitrite occurred in the roots only in small amounts and the ratio of activities of these isoenzymes was not affected significantly. NADP⁺-GDH activity, detected in the gels at the same site as NAD⁺-GDH₁, was depressed by increasing nitrite concentrations.     

Alteration of substrate specificity of valine dehydrogenase from Streptomyces albus

The catabolism of branched chain amino acids, especially valine, appears to play an important role in furnishing building blocks for macrolide and polyether antibiotic biosyntheses. To determine the active site residues of ValDH, we previously cloned, partially characterized, and identified the active site (lysine) of Streptomyces albus ValDH. Here we report further characterization of S. albus ValDH. The molecular weight of S. albus ValDH was determined to be 38 kDa by SDS-PAGE and 67 kDa by gel filtration chromatography indicating that the enzyme is composed of two identical subunits. Optimal pHs were 10.5 and 8.0 for dehydrogenase activity with valine and for reductive amination activity with -ketoisovaleric acid, respectively. Several chemical reagents, which modify amino-acid side chains, inhibited the enzyme activity. To examine the role played by the residue for enzyme specificity, we constructed mutant ValDH by substituting alanine for glycine at position 124 by site-directed mutagenesis. This residue was chosen because it has been considered to be important for substrate discrimination by phenylalanine dehydrogenase (PheDH) and leucine dehydrogenase (LeuDH). The Ala-124–Gly mutant enzyme displayed lower activities toward aliphatic amino acids, but higher activities toward L-phenylalanine, L-tyrosine, and L-methionine compared to the wild type enzyme suggesting that Ala-124 is involved in substrate binding in S. albus ValDH.     

The structure and allosteric regulation of glutamate dehydrogenase

Glutamate dehydrogenase (GDH) has been extensively studied for more than 50 years. Of particular interest is the fact that, while considered by most to be a ‘housekeeping’ enzyme, the animal form of GDH is heavily regulated by a wide array of allosteric effectors and exhibits extensive inter-subunit communication. While the chemical mechanism for GDH has remained unchanged through epochs of evolution, it was not clear how or why animals needed to evolve such a finely tuned form of this enzyme. As reviewed here, recent studies have begun to elucidate these issues. Allosteric regulation first appears in the Ciliates and may have arisen to accommodate evolutionary changes in organelle function. The occurrence of allosteric regulation appears to be coincident with the formation of an ‘antenna’ like feature rising off the tops of the subunits that may be necessary to facilitate regulation. In animals, this regulation further evolved as GDH became integrated into a number of other regulatory pathways. In particular, mutations in GDH that abrogate GTP inhibition result in dangerously high serum levels of insulin and ammonium. Therefore, allosteric regulation of GDH plays an important role in insulin homeostasis. Finally, several compounds have been identified that block GDH-mediated insulin secretion that may be to not only find use in treating these insulin disorders but to kill tumors that require glutamine metabolism for cellular energy.     

Altering the substrate specificity of glutamate dehydrogenase from Bacillus subtilis by site-directed mutagenesis

The Lys80, Gly82 and Met101 residues of glutamate dehydrogenase from Bacillus subtilis were mutated into a series of single mutants. The wild-type enzyme was highly specific for 2-oxoglutarate, whereas G82K and M101S dramatically switched to increased specificity for oxaloacetate with kcat values 3.45 and 5.68 s-1, which were 265-fold and 473-fold higher respectively than those for 2-oxoglutarate.     

Characterization of glutamate dehydrogenase immobilization on silica surface by atomic force microscopy and kinetic analyses

Covalent immobilization of glutamate dehydrogenase (GDH) onto activated Si/SiO₂ supports was analyzed by both atomic force microscopy (AFM) and an enzymatic assay. When the concentration of 3-aminopropyltriethoxysilane used in the first derivatization step of the silicon surface was decreased, the specific enzymatic activity also decreased, whereas the mean roughness increased. Thus, the activity of immobilized GDH is critically dependent on the conditions for surface derivatization, and is inversely correlated with surface roughness.     

Printing of polymer microcapsules for enzyme immobilization on paper substrate

Poly(ethyleneimine) (PEI) microcapsules containing laccase from Trametes hirsuta (ThL) and Trametes versicolor (TvL) were printed onto paper substrate by three different methods: screen printing, rod coating, and flexo printing. Microcapsules were fabricated via interfacial polycondensation of PEI with the cross-linker sebacoyl chloride, incorporated into an ink, and printed or coated on the paper substrate. The same ink components were used for three printing methods, and it was found that laccase microcapsules were compatible with the ink. Enzymatic activity of microencapsulated TvL was maintained constant in polymer-based ink for at least eight weeks. Thick layers with high enzymatic activity were obtained when laccase-containing microcapsules were screen printed on paper substrate. Flexo printed bioactive paper showed very low activity, since by using this printing method the paper surface was not fully covered by enzyme microcapsules. Finally, screen printing provided a bioactive paper with high water-resistance and the highest enzyme lifetime.     

The structure and allosteric regulation of mammalian glutamate dehydrogenase

Glutamate dehydrogenase (GDH) is a homohexameric enzyme that catalyzes the reversible oxidative deamination of l-glutamate to 2-oxoglutarate. Only in the animal kingdom is this enzyme heavily allosterically regulated by a wide array of metabolites. The major activators are ADP and leucine, while the most important inhibitors include GTP, palmitoyl CoA, and ATP. Recently, spontaneous mutations in the GTP inhibitory site that lead to the hyperinsulinism/hyperammonemia (HHS) syndrome have shed light as to why mammalian GDH is so tightly regulated. Patients with HHS exhibit hypersecretion of insulin upon consumption of protein and concomitantly extremely high levels of ammonium in the serum. The atomic structures of four new inhibitors complexed with GDH complexes have identified three different allosteric binding sites. Using a transgenic mouse model expressing the human HHS form of GDH, at least three of these compounds were found to block the dysregulated form of GDH in pancreatic tissue. EGCG from green tea prevented the hyper-response to amino acids in whole animals and improved basal serum glucose levels. The atomic structure of the ECG-GDH complex and mutagenesis studies is directing structure-based drug design using these polyphenols as a base scaffold. In addition, all of these allosteric inhibitors are elucidating the atomic mechanisms of allostery in this complex enzyme.     

Thermodynamic regulation of human short-chain acyl-CoA dehydrogenase by substrate and product binding

Human short-chain acyl-CoA dehydrogenase (hSCAD) catalyzes the first matrix step in the mitochondrial beta-oxidation cycle for substrates with four and six carbons. Previous studies have shown that the act of substrate/product binding induces a large enzyme potential shift in acyl-CoA dehydrogenases. The objective of this work was to examine the thermodynamic regulation of this process through direct characterization of the electrochemical properties of hSCAD using spectroelectrochemical methodology. A large amount of substrate activation was observed in the enzymatic reaction of hSCAD (+33 mV), the greatest magnitude measured in any acyl-CoA dehydrogenase to date. To examine the role of the substrate as well as the product in electron transfer by hSCAD, a catalytic base mutation (E368Q) was constructed. The E368Q mutation inactivates the reductive and oxidative pathways such that the individual effects of substrate and product binding on the redox potential can be investigated. Optimal substrate (butyryl-CoA) was seen to shift the flavin redox potential slightly more positive (+38 mV) than did optimal product (crotonyl-CoA) (+31 mV), a finding opposite of that observed in another short-chain enzyme, bacterial SCAD. These results indicate that substrate redox activation occurs in hSCAD leading to a large enzyme midpoint potential shift. Substrate binding in hSCAD appears to make a larger contribution than does product to thermodynamic modulation.     

Alteration in substrate specificity of horse liver alcohol dehydrogenase by an acyclic nicotinamide analog of NAD+

A new, acyclic NAD-analog, acycloNAD⁺ has been synthesized where the nicotinamide ribosyl moiety has been replaced by the nicotinamide (2-hydroxyethoxy)methyl moiety. The chemical properties of this analog are comparable to those of β-NAD⁺ with a redox potential of −324 mV and a 341 nm λ_max for the reduced form. Both yeast alcohol dehydrogenase (YADH) and horse liver alcohol dehydrogenase (HLADH) catalyze the reduction of acycloNAD⁺ by primary alcohols. With HLADH 1-butanol has the highest V_max at 49% that of β-NAD⁺. The primary deuterium kinetic isotope effect is greater than 3 indicating a significant contribution to the rate limiting step from cleavage of the carbon–hydrogen bond. The stereochemistry of the hydride transfer in the oxidation of stereospecifically deuterium labeled n-butanol is identical to that for the reaction with β-NAD⁺. In contrast to the activity toward primary alcohols there is no detectable reduction of acycloNAD⁺ by secondary alcohols with HLADH although these alcohols serve as competitive inhibitors. The net effect is that acycloNAD⁺ has converted horse liver ADH from a broad spectrum alcohol dehydrogenase, capable of utilizing either primary or secondary alcohols, into an exclusively primary alcohol dehydrogenase. This is the first example of an NAD analog that alters the substrate specificity of a dehydrogenase and, like site-directed mutagenesis of proteins, establishes that modifications of the coenzyme distance from the active site can be used to alter enzyme function and substrate specificity. These and other results, including the activity with α-NADH, clearly demonstrate the promiscuity of the binding interactions between dehydrogenases and the riboside phosphate of the nicotinamide moiety, thus greatly expanding the possibilities for the design of analogs and inhibitors of specific dehydrogenases.