The oxidation state of active site thiols determines activity of saccharopine dehydrogenase at low pH

Saccharopine dehydrogenase catalyzes the NAD-dependent conversion of saccharopine to generate l-lysine and α-ketoglutarate. A disulfide bond between cysteine 205 and cysteine 249, in the vicinity of the dinucleotide-binding site, is observed in structures of the apoenzyme, while a dithiol is observed in a structure with AMP bound, suggesting preferential binding of the dinucleotide to reduced enzyme. Mutation of C205 to S gave increased values of V/E_t and V/KE_t at pH 7 compared to wild type. Primary deuterium and solvent deuterium kinetic isotope effects suggest the catalytic pathway, which includes the hydride transfer and hydrolysis steps, contributes more to rate limitation in C205S, but the rates of the two steps relative to one another remain the same. There is a large increase in the rate constants V₁/E_t and V₁/K_NADEt at pH values below 7 compared to WT. Data indicate the low pH increase in activity results from a decreased sensitivity of the C205S mutant enzyme to the protonation state of an enzyme group with a pK_a of about 7, likely responsible for a pH-dependent conformational change. Reduction of WT and C205S mutant enzymes with TCEP gives equal activities at pH 6, consistent with the increased activity observed for the C205S mutant enzyme.     

Purification and properties of alpha-ketoadipate reductase, a newly discovered enzyme from human placenta.

A newly discovered enzyme, α-ketoadipate reductase, has been purified 1000-fold from human placenta. This enzyme catalyzes the following reaction: α-ketoadipate + NADH + H⁺ → α-hydroxyadipate + NAD. The enzyme has an estimated molecular weight of 95,000 on gel filtration and an isoelectric point at pH 7.0 on electrofocusing. Several forms of the enzyme were isolated during purification. The pH optimum for the major form was 6.3. The reaction product of α-ketoadipate reductase was identified as α-hydroxyadipate by comparison of the enzyme product with chemically prepared α-hydroxyadipate. Studies of the reaction stoichiometry indicated that equimolar quantities of NADH and α-ketoadipate were used in the synthesis of an equivalent quantity of α-hydroxyadipate. Under conditions where the remaining lactate dehydrogenase and malate dehydrogenase were completely inhibited without affecting the α-ketoadipate reductase activity, it was found that α-ketoadipate reductase was highly specific for α-ketoadipate as substrate. NADPH could not substitute for NADH. Initial velocity experiments showed that NADH was an uncompetitive substrate inhibitor.     

Anomalous “saturation kinetics” occurring in the reaction of l-α-aminoadipate with pig heart aspartate aminotransferase

Low concentrations of l-α-aminoadipate and α-ketoadipate do not form stable complexes with the phosphopyridoxal and phosphopyridoxamine forms of pig heart aspartate aminotransferase, respectively, as judged by direct spectroscopic analyses. Furthermore, the addition of 40 mm aminoadipate did not significantly inhibit the transamination reaction between aspartate and α-ketoglutarate. Nevertheless, aminoadipate complexes must be formed, albeit in very small amounts, because the phosphopyridoxal form of the enzyme undergoes transamination with aminoadipate to form the phosphopyridoxamine form and α-ketoadipate. Since the reaction of aminoadipate with the phosphopyridoxal form of the enzyme shows apparent saturation kinetics at substrate concentrations that are insufficient to form detectable intermediary complexes, the rate-limiting step in the reaction must be a slow transition of the enzyme occurring prior to the amino acid interaction. The maximum rate of this transamination reaction decreased with an increase in the buffer anion concentration; the anion must, therefore, inhibit the slow enzyme transition. The experimental data are consistent with a simple model in which the anion dissociates before the slow transition that allows the aminoadipate to react. The turnover rate of aminoadipate is approximately three orders of magnitude less than those of both aspartate and glutamate. The latter substrates do not appear, therefore, to require the slow enzyme transition that is necessary for aminoadipate to react.     

A Branched-chain Amino Acid Aminotransferase from the Rumen Ciliate Genus Entodinium

A branched-chain amino acid aminotransferase was extracted from rumen ciliates of the genus Entodinium and was partially purified by Sephadex G-200, DEAE-cellulose and DEAE-Sephadex A-50 column chromatography. The purified enzyme was active only with leucine, isoleucine and valine, and required pyridoxal phosphate as cofactor. The amino acids competed with each other as substrates. The enzyme had optimal activity at pH 6.0 in phosphate buffer. The K_m values for the substrates and cofactor are as follows: 1.66 for leucine; 0.90 for isoleucine; 0.79 for valine; 0.29 mM for α-ketoglutarate: and 0.1 μM for pyridoxal phosphate. Enzyme activity was inhibited by p-chloromercuribenzoate and HgCl₂. Gel filtration indicated the enzyme to have a molecular weight of 34,000.     

The conversion of L-lysine to saccharopine and alpha-aminoadipate in mouse

Saccharopine [?-N-(l-glutaryl-2)-l-lysine] has been found to occur in normal, untreated mouse liver. The pool of saccharopine as well as that of α-aminoadipate become labeled shortly after the administration of l-lysine-U-¹⁴C into intact mouse. In vitro experiments using the mouse liver homogenate have shown that l-lysine is converted to saccharopine in the presence of α-ketoglutarate and NADPH, and saccharopine to α-aminoadipate in the presence of NAD⁺. The oxidation of α-aminoadipic-δ-semialdehyde (Δ¹-piperideine-6-carboxylate), the proposed reaction product of saccharopine cleavage, to α-aminoadipate is effected by either NAD⁺ or NADP⁺.     

Determination of plasma and serum l-lysine using l-lysine ε-oxidase from Marinomonas mediterranea NBRC 103028

This article describes a successful application of l-lysine ε-oxidase (EC 1.4.3.20) for l-lysine determination. l-Lysine ε-oxidase was isolated from culture supernatant of Marinomonas mediterranea NBRC 103028^T and was used for l-lysine determination. Comparison of the characteristics of l-lysine ε-oxidase with l-lysine α-oxidase, a commercial enzyme used for l-lysine determination, suggests that the use of l-lysine ε-oxidase would be more valuable for the determination of l-lysine because of its selectivity and sensitivity, especially in samples with low l-lysine concentration. The enzyme acted only on l-lysine and l-ornithine, to which the relative activity was only 3.4% of that on l-lysine. The value obtained by the colorimetric assay using l-lysine ε-oxidase and horseradish peroxidase was not affected by l-ornithine. The enzyme also shows a higher affinity for l-lysine (K_m = 0.0018 mM). l-Lysine determination using l-lysine ε-oxidase in human plasma and serum was examined. The measured values were close to values determined by instrumental analyses using the precolumn AccQ·Tag Ultra Derivatization Kit. These results suggest that l-lysine ε-oxidase can be used for diagnosis based on plasma l-lysine concentration. This is the first report on the application of l-lysine ε-oxidase.     

Purification and Properties of α-Aminoadipate Aminotransferase from Rat Liver and Kidney Mitochondria

Abstract

Recently we reported an affinity chromatography method to purify α-aminoadipate aminotransferase (AadAT) activity from rat kidney supernatant fraction. Using the same affinity column, we purified AadAT activities from rat kidney and liver mitochondria. The physical and kinetic properties such as pH optima, Km for substrates, molecular weight, subunit structure, isoelectric pH, electrophoretic mobility and inhibition by dicarboxylic acids of mitochondrial AadAT were similar to those of the AadAT from rat kidney supernatant fraction. These results indicate that AadAT from different subcellular fractions is structurally and immunologically identical.     

One-electron reduction of adriamycin: Properties of the semiquinone

Pulse radiolysis of aqueous solutions containing adriamycin and redox indicators of known one-electron reduction potential (E¹) shows that its E¹ at pH 7 is ?328 mV (vs NHE). The variation E¹ with pH in the range 6–12 shows that the net charge on the semiquinone at pH 7 is zero. As well as the pK_a values of 2.9 and ≥ 14 established independently, the semiquinone has a pK_a close to 9.2. The new data enable the structure and likely reactivity of the semiquinone to be specified.     

Spectroscopic Analysis of the Weak Light Generated in Autoxidation of Linseed Oil

Nucleoside oxidase purified from Pseudomonas maltophilia LB-86 had mol. wt. = 130.000 and was composed of one each of four non-identical subunits: subunit α, 76,000; subunit β, 33,000; subunit γ, 18,000; subunit δ, 14,000. The enzyme contains 1 mol of covalently bound FAD, 2g atoms of nonheme iron, 2 mol of labile sulfides, and 1 mol of heme per mol enzyme protein. The absorption spectrum of nucleoside oxidase had maxima 278 and 390 nm, and shoulders at 343 and 450 nm.

The enzyme catalyzes the oxidation of various nucleosides, and the Km value for inosine was 4.4 × 10^-5 M. The enzyme was most active at pH 5 ~ 6, and was most stable between pH 5.0 ~ 6.0 and at temperatures below 60°C. The activity was strongly inhibited by N-bromosuccinimide and potassium cyanide.     

通过易错PCR提高鼠伤寒沙门氏菌丙氨酸消旋酶催化活性

[目的] 通过易错PCR技术提高鼠伤寒沙门氏菌中丙氨酸消旋酶的催化活性。[方法] 利用易错PCR技术构建丙氨酸消旋酶基因alr_St的突变体文库,采用缺陷菌株UT5028筛选突变体基因,以D-氨基酸氧化酶偶联法检测各突变蛋白的活性,通过凝胶过滤层析法分析酶蛋白寡聚化状态,并采用HPLC检测酶蛋白的动力学参数。[结果] 经过易错PCR及定点突变技术最终获得了3个催化活性有所提高的突变体A3V、Y343H和A3VY343H,酶学特性分析发现,与野生型蛋白StAlr相比,突变体Y343H仅对底物L/D-丝氨酸的催化效率略有提高,k_cat/K_m值分别是StAlr的2.01和3.68倍;而突变体A3V则对底物L/D-丙氨酸或L/D-丝氨酸的K_m、k_cat和k_cat/K_m值均有较大幅度的改变,其k_cat/K_m值分别是StAlr的105.51、97.36、4.63和10.73倍。凝胶过滤层析结果显示,突变体A3V在蛋白含量极低时就呈现出单体和二聚体共存状态,且随着蛋白含量的增加,其向二聚体状态迁移的速率最为明显。[结论] 丙氨酸消旋酶StAlr的第3位点是影响其催化活性和低聚合状态的关键位点。     

Immobilization of Bacillus subtilis α-amylase on zirconia-coated alkylamine glass with glutaraldehyde

Bacillus subtilis α-amylase (EC 3.2.1.1) has been immobilized on zirconia-coated alkylamine glass by using the process of glutaraldehyde coupling. The immobilized enzyme preparation exhibited 52% of the initial enzyme activity and a conjugation yield of 28 mg/g support. The K_m value of the immobilized α-amylase was decreased by immobilization while V_max was unaltered. E_a of the enzyme was decreased upon conjugation. The soluble enzyme was optimally active at pH 5.6 while the immobilized enzyme exhibited optimal activity in the pH range 5.4–6.2. The alkylamine-immobilized enzyme has also been characterized through its isoelectric point. The industrial importance of this work is discussed.     

Relevance of phenylalanine 216 in the affinity of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> phosphoenolpyruvate carboxykinase for Mn(II)

Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase catalyzes the reversible formation of oxaloacetate and adenosine triphosphate from PEP, adenosine diphosphate and carbon dioxide, and uses Mn²⁺ as the activating metal ion. Comparison with the crystalline structure of homologous Escherichia coli PEP carboxykinase [Tari et al. (1997) Nature Struct. Biol. 4, 990–994] shows that Lys²¹³ is one of the ligands to Mn²⁺ at the enzyme active site. Coordination of Mn²⁺ to a lysyl residue is not common and suggests a low pK _a value for the ε-NH₂ group of Lys²¹³. In this work, we evaluate the role of neighboring Phe²¹⁶ in contributing to provide a low polarity microenvironment suitable to keep the ε-NH₂ of Lys²¹³ in the unprotonated form. Mutation Phe216Tyr shows that the introduction of a hydroxyl group in the lateral chain of the residue produces a substantial loss in the enzyme affinity for Mn²⁺, suggesting an increase of the pK _a of Lys²¹³. In agreement with this interpretation, theoretical calculations indicate an alkaline shift of 2.8 pH units in the pK _a of the ε-amino group of Lys²¹³ upon Phe216Tyr mutation.     

Effects of pH on benzimidazole fluorescence

Four benzimidazoles (unsubstituted, 5-methyl, 2-ethyl, and 2-ethyl-5-methyl) have been characterized by fluorescence spectroscopy. At low pH (<6), activation at 270 nm caused fluorescence at 305 nm; at high pH (<8), activation at 270 nm caused fluorescence at 365 nm. The relative proportion of peak fluorescence at either 305 or 365 nm was correlated with the pK_a values of the four benzimidazoles. It was concluded that the protonated specie of benzimidazole was fluorescent at 365 nm and the unprotonated specie was also fluorescent at 305 nm.     

Identification,purification and partial characterization of a 70 kDa inhibitor protein of Na+/K+-ATPase from cytosol of pulmonary artery smooth muscle

AimsWe sought to identify, purify and partially characterize a protein inhibitor of Na⁺/K⁺-ATPase in cytosol of pulmonary artery smooth muscle.Main methods(i) By spectrophotometric assay, we identified an inhibitor of Na⁺/K⁺-ATPase in cytosolic fraction of pulmonary artery smooth muscle; (ii) the inhibitor was purified by a combination of ammonium sulfate precipitation, diethylaminoethyl (DEAE) cellulose chromatography, hydroxyapatite chromatography and gel filtration chromatography; (iii) additionally, we have also purified Na⁺/K⁺-ATPase α₂β₁ and α₁β₁ isozymes for determining some characteristics of the inhibitor.Key findingsWe identified a novel endogenous protein inhibitor of Na⁺/K⁺-ATPase having an apparent mol mass of ～ 70 kDa in the cytosolic fraction of the smooth muscle. The IC₅₀ value of the inhibitor towards the enzyme was determined to be in the nanomolar range. Important characteristics of the inhibitor are as follows: (i) it showed different affinities toward the α₂β₁ and α₁β₁ isozymes of the Na⁺/K⁺-ATPase; (ii) it interacted reversibly to the E₁ site of the enzyme; (iii) the inhibitor blocked the phosphorylated intermediate formation; and (iv) it competitively inhibited the enzyme with respect to ATP. CD studies indicated that the inhibitor causes an alteration of the conformation of the enzyme. The inhibition study also suggested that the DHPC solubilized Na⁺/K⁺-ATPase exists as (αβ)₂ diprotomer.SignificanceThe inhibitor binds to the Na⁺/K⁺-ATPase at a site different from the ouabain binding site. The novelty of the inhibitor is that it acts in an isoform specific manner on the enzyme, where α₂ is more sensitive than α₁.     

Structural-based screening of L-phenylglycine aminotransferase using L-phenylalanine as the optimal amino donor: Recycling of L-phenylalanine to produce L-phenylglycine

The aproteinogenic amino acid, L-phenylglycine, is an important side chain building block for some drugs. It would be of great commercial and environmental value to biocatalyse L-phenylalanine to L-phenylglycine, and thus replace the organic synthesis method. To produce L-phenylglycine from L-phenylalanine, an L-phenylglycine aminotransferase was screened and characterized. HpgT_AO showed high homology to α-aminoadipate aminotransferase. The L-phenylalanine binding site was near the residues S26, R401, N201, and G46 in HpgT_AO, and L-phenylalanine formed a hydrogen bond with Asn20, which was similar to the substrate binding mechanism of α-aminoadipate aminotransferase. HpgT_AO showed increased activity in alkalescent environment below 40°C. The kinetic analysis showed that L-phenylalanine had the highest affinity to HpgT_AO, which ensured the recycle biosynthesis of Lphenylglycine from L-phenylalanine. To date, it was the only aminotransferase using L-phenylalanine as an optimal amino donor. The L-phenylglycine biocatalysis operon was also constructed by co-expressing the hmaS, hmo and hpgT by a single plasmid. The first in vitro conversion of L-phenylalanine to L-phenylglycine was achieved by directly using the L-phenylalanine fermentation broth as the raw material.     

Porcine cytosolic aspartate aminotransferase reconstituted with [4'-13C]pyridoxal phosphate. pH- and ligand-induced changes of the coenzyme observed by 13C NMR spectroscopy

Apoenzyme samples of aspartate aminotransferase (AspAT) purified from the cytosolic fraction of pig heart were reconstituted with [4'-13C]pyridoxal 5'-phosphate (pyridoxal-P). The 13C NMR spectra of AspAT samples thus generated established the chemical shift of 165.3 ppm for C4' of the coenzyme bound as an internal aldimine with lysine 258 of the enzyme at pH 5. In the absence of ligands the chemical shift of C4' was shown to be pH dependent, shifting 5 ppm upfield to a constant value of 160.2 ppm above pH 8, the resulting pKa of 6.3 in agreement with spectrophotometric titrations. The addition of the competitive inhibitor succinate to the internal aldimine raises the pKa of the imine to 7.8, consistent with the theory of charge neutralization in the active site. In the presence of saturating concentrations of 2-methylaspartic acid the C4' signal of the coenzyme was shown to be invariant with pH and located at 162.7 ppm, midway between the observed chemical shifts of the protonated and unprotonated forms of the internal aldimine. The intermediate chemical shift of the external aldimine complex is thought to reflect the observation of an equilibrium mixture composed of roughly equal populations of the protonated ketoenamine and a dipolar anion species, corresponding to their respective spectral bands at 430 and 360-370 nm. Conversion to the pyridoxamine form was accomplished via reaction of the internal aldimine with L-cysteinesulfinate or by reduction with sodium borohydride, and the resulting C4' chemical shifts were identified by difference spectroscopy. Finally, the line widths of the C4' resonance under the various conditions were measured and qualitatively compared. The results are discussed in terms of the current mechanism and molecular models of the active site of AspAT.     

INHIBITION OF ALANINE AND ASPARTATE AMINOTRANSFERASES BY α-OXODERIVATIVES OF THE BRANCHED-CHAIN AMINO ACIDS

Abstract—

• 1 L-Alanine: α-oxoglutarate aminotransferase was partly purified from rat brain and liver. The enzyme from the brain has about 10 times less activity than that from the liver.
• 2 Both enzymes have identical apparent K_m values for L-alanine, L-glutamate, α-oxoglutarate and pyruvate. Moreover they are competitively inhibited by L-leucine. α-oxoisocaproate and α-oxotsovalerate. Obtained K, values are very similar and do not depend on the course of reaction.
• 3 α-Oxoisocaproate inhibits the activity of crystalline L-aspartate: α-oxoglutarate aminotransferase; Kj is about 4–7 mM.
• 4 The pyridoxamine form of L-alanine: α-oxoglutarate aminotransferase seems to be more sensitive to the inhibitory effect of the compounds investigated.
• 5 The effect of branched-chain amino acids and their α-oxoanalogues on the metabolism of amino groups in maple syrup urine disease is discussed.

     

Phenylalanine- and tyrosine-auxotrophic mutants of Saccharomyces cerevisiae impaired in transamination

This paper reports the first isolation of Saccharomyces cerevisiae mutants lacking aromatic aminotransferase I activity (aro8), and of aro8 aro9 double mutants which are auxotrophic for both phenylalanine and tyrosine, because the second mutation, aro9, affects aromatic aminotransferase II. Neither of the single mutants displays any nutritional requirement on minimal ammonia medium. In vitro, aromatic aminotransferase I is active not only with the aromatic amino acids, but also with methionine, α-aminoadipate, and leucine when phenylpyruvate is the amino acceptor, and in the reverse reactions with their oxo-acid analogues and phenylalanine as the amino donor. Its contribution amounts to half of the glutamate:2-oxoadipate activity detected in cell-free extracts and the enzyme might be identical to one of the two known α-aminoadipate aminotransferases. Aromatic aminotransferase I has properties of a general aminotransferase which, like several aminotransferases of Escherichia coli, may be able to play a role in several otherwise unrelated metabolic pathways. Aromatic aminotransferase II also has a broader substrate specificity than initially described. In particular, it is responsible for all the measured kynurenine aminotransferase activity. Mutants lacking this activity grow very slowly on kynurenine medium.     

Purification of branched-chain amino acid aminotransferase from <Emphasis Type="Italic">Helicobacter pylori</Emphasis> NCTC 11637

Summary. Branched-chain amino acid aminotransferase was purified by several column chromatographies from Helicobacter pylori NCTC 11637, and the N-terminal amino acid sequence was analyzed. The enzyme gene was sequenced based on a putative branched-chain amino acid aminotransferase gene, ilvE of H. pylori 26695, and the whole amino acid sequence was deduced from the nucleotide sequence. The enzyme existed in a homodimer with a calculated subunit molecular weight (MW) of 37,539 and an isoelectric point (pI) of 6.47. The enzyme showed high affinity to 2-oxoglutarate (K _m = 0.085 mM) and L-isoleucine (K _m = 0.34 mM), and V _max was 27.3 μmol/min/mg. The best substrate was found to be L-isoleucine followed by L-leucine and L-valine. No activity was shown toward the D-enantiomers of these amino acids. The optimal pH and temperature were pH 8.0 and 37 °C, respectively.     

Synthesis of the segment (11–23) located in the first tandem repeat of plasma kallikrein: comparative binding studies of this and another segment (328–343) to high-molecular-mass kininogen

The synthesis of porcine plasma kallikrein (pPK) segment (11–23), of sequence Phe-Phe-Arg-Gly-Gly-Asp-Val-Ser-Ala-Met-Tyr-Thr-Pro, present in the first tandem repeat sequence of the regulatory chain of PK, has been accomplished following the peptide fragments (5 + 4 + 4) condensation strategy in solution, as well as by fluorenylmethoxycarbonyl solid-phase chemistry. This and another synthetic PK segment of residues (328–343) present in the fourth tandem repeat sequence [Cys(ACM)-Ser-Leu-Arg-Leu-Ser-Thr-Asp-Gly-Ser-Pro-Thr-Arg-Ile-Thr-Tyr] and synthesized by a solid-phase method, were fully characterized by ¹H nuclear magnetic resonance, fast atom bombardment mass spectrometry, amino acid composition and reversed-phase high-performance liquid chromatography. Proteolysis of these peptides by either rat PK (rPK) or trypsin resulted in cleavages between Arg↓Gly for pPK (11–23) and between Arg↓Leu and Arg↓Ile for rPK (328–343). Kinetic studies revealed that for peptide pPK (11–23), the catalytic efficiency (k_cat/K_m) of rPK is 9-fold higher than that of trypsin, but for the other peptide, rPK (328–343), k_cat/K_m of trypsin is 49-fold higher than that of rPK. The facile cleavage of pPK (11–23) by rPK confirms the Arg¹³↓Gly¹⁴ position as the site of autolytic degradation of PK and also explains its special preference for Phe-Phe-Arg sequence.