Thermolabile Enzyme Research Articles

Purification and characterization of 2,4-dichlorophenoxyacetate/alpha-ketoglutarate dioxygenase.

The Alcaligenes eutrophus 2,4-dichlorophenoxyacetate/alpha-ketoglutarate dioxygenase, encoded by the tfdA gene of plasmid pJP4, is an Fe(II)-dependent enzyme that catalyzes the conversion of 2,4-dichlorophenoxyacetate to 2,4-dichlorophenol and glyoxylate concomitant with the decomposition of alpha-ketoglutarate to form succinate and carbon dioxide (Fukumori, F., and Hausinger, R. P. (1993) J. Bacteriol. 175, 2083-2086). Using recombinant Escherichia coli cells that overexpress the tfdA gene, the thermolabile enzyme (stable only up to 30 degrees C) was purified to apparent homogeneity (specific activity of 16.9 mumol of substrate converted min-1 mg of protein-1) by a simple two-step procedure. The native protein has an apparent M(r) of 50,000 +/- 2,500, consistent with a homodimeric structure. Ferrous ion is absolutely required for activity and cannot be replaced by several other divalent cations tested. Ascorbic acid stimulates dioxygenase activity and reduces the rate of enzyme inactivation by a metal ion-mediated process. The enzyme exhibits maximum activity at pH 6.5-7, however, it is stable over a pH range of 6.5-11. Although capable of hydroxylating a wide range of phenoxyacetates and related compounds, the enzyme exhibits the greatest affinity (Km 17.5 +/- 1.0 microM) and highest catalytic efficiency for 2,4-dichlorophenoxyacetate. Similarly, alpha-ketoglutarate is the preferred co-substrate (Km 3.20 +/- 0.54 microM) for the enzyme, but it can utilize a range of other alpha-ketoacids with lower efficiency. Results from chemical modification studies are consistent with the presence of multiple essential histidine residues in the enzyme.

Amino acid residues involved in the catalytic site of human erythrocyte bisphosphoglycerate mutase. Functional consequences of substitutions of His10, His187 and Arg89.

Human bisphosphoglycerate mutase (GriP2 mutase) is a trifunctional enzyme which synthesizes and degrades GriP2 in red cells. Among the amino acid residues involved in its active site there are two conserved histidine residues, His10 which is phosphorylated during the catalytic process and His187 for which only speculative data have been made about the potential role during the reactions. Another amino acid residue, Arg89, had not been described as part of this active site but we have recently shown that a natural mutant Arg89-->Cys was highly thermolabile and showed severe perturbations of its enzymatic properties. To understand better the exact role of these residues, replacements of His10 by Gly (H10G) or Asp (H10D), His187 by Asn (H187N), Tyr (H187Y) or Asp (H187D) and Arg89 by Cys (R89C), Ser (R89S), Gly (R89G) or Lys (R89K) were performed by site-directed mutagenesis. The results obtained in this report show that replacement of the His10 residue completely abolished the enzymatic activities. Concerning the His187 residue, our results afford arguments that it plays an essential role in the three catalytic activities. Indeed all these activities are abolished in the two H187Y and H187D variants, whereas they are detectable though strongly diminished, for the H187N variant. In addition mutations at His187 could be distinguishable from those at His10 since the former resulted in a thermolabile enzyme, whereas no significant change in heat stability was observed for the latter. It is noteworthy that the H187N variant is protected against thermal instability by glycerate 2,3-bisphosphate (GriP2). Concerning the Arg89 mutants, R89C, R89S and R89G, the three variants showed characteristics identical to those found in the natural R89C mutant, i.e. loss of 99% of synthase activity, consistent decrease of mutase and 2-phosphoglycolate-stimulated phosphatase activities whereas the unstimulated phosphatase activity was normal. Moreover these mutants were unstable at 55 degrees C but GriP2 was able to protect them against thermal instability. In contrast, the R89K mutant was stable at 55 degrees C. Its synthase and unstimulated phosphatase activities were normal but its mutase and 2-phosphoglycolate-stimulated phosphatase activities were decreased. In addition, Km values for monophosphoglycerates were increased (3.2-fold) in the synthase but normal in mutase activities, whereas Km values for GriP2 were normal in mutase and phosphatase activities.(ABSTRACT TRUNCATED AT 400 WORDS)

Hyperthermostable variants of a highly thermostable alpha-amylase.

Genetic screening at temperatures between 70-80 degrees C far exceeds the range of growth of most bacteria, and is not applicable to isolate easily thermostable protein variants. We describe a temperature shift protocol and an in vivo screening method which allowed us to identify a hyperthermostable variant of the thermostable alpha-amylase from Bacillus licheniformis. Our strategy was to select, after hydroxylamine mutagenesis, an intragenic suppressor mutation which overcomes a mutation leading to a thermolabile enzyme. Sequence analysis of the mutated gene revealed only one change in the amino acid sequence, substituting a valine for alanine at position 209. This single amino acid replacement increased the half-life of the protein at 90 degrees C by a factor of two to three relative to the wild-type enzyme. When this substitution was combined with another stabilizing substitution (H133Y) we described previously, the stabilizing effects were additive. The half-life of the new protein was about 12 hours at 90 degrees C, corresponding to a nine to ten-fold increase over the wild-type enzyme and the industrial Bacillus licheniformis alpha-amylase Termamyl. These mutations are located in a predicted folding domain of the protein which appears crucial in determining thermal stability.

Human triosephosphate isomerase: substitution of Arg for Gly at position 122 in a thermolabile electromorph variant, TPI-Manchester.

Denaturing gradient gel electrophoreses of polymerase chain reaction amplified DNA products and subsequent direct sequencing identified a G-to-A transition causing a replacement of Gly 122 with Arg in an electrophoretic mobility variant of human triosephosphate isomerase, TPI-Manchester. This was the only TPI electromorph variant detected in screening of greater than 3,400 humans in an Ann Arbor, Mich. population. This substitution is at the amino terminus or solvent interaction end of the fifth beta sheet of the alpha/beta barrel structure. The TPI-Manchester variant is a thermolabile enzyme, but the stability of the variant enzyme is not sensitive to other denaturants. This amino acid substitution does not involve residues of the active site and does not detectably alter the kinetic properties of the enzyme. The data provide additional insight into the amino acid residues that are important for the maintenance of the structural characteristics of this very evolutionary constrained protein.

Effect of Age on Thermal Inactivation of Glucose-6-Phosphate Dehydrogenase in Liver and Kidney of the Male Garden Lizard

A comparison between middle-aged and old lizards revealed no significant age change in glucose-6-phosphate dehydrogenase activity of liver and kidney. Thermal inactivation at 51 +/- 1 degree C induced a significantly higher degree of decline in enzyme activity of tissues of old than that of middle-aged lizards. The increase in thermolability of enzyme in liver and kidney of ageing lizards suggests an accumulation of altered enzyme molecules.

Human liver catechol-O-methyltransferase pharmacogenetics

Catechol-O-methyltransferase activity and thermal stability in the human red blood cell are controlled by a common genetic polymorphism. Approximately 25% to 30% of a randomly selected population sample is homozygous for the traits of low catechol-O-methyltransferase activity and thermolabile enzyme in the red blood cell. We tested the hypothesis that the catechol-O-methyltransferase genetic polymorphism might also control those same characteristics of the enzyme in an important human drug-metabolizing organ, the liver. Catechol-O-methyltransferase enzyme activity and thermal stability were measured in 99 hepatic biopsy samples obtained during clinically indicated surgery. The frequency distribution of heated/control ratios, a measure of enzyme thermal stability, was bimodal, with 28% of samples included in a subgroup with thermolabile enzyme. There were no sex-related differences in hepatic catechol-O-methyltransferase thermal stability. However, catechol-O-methyltransferase enzyme activity in hepatic tissue from male subjects was significantly higher than that in samples from female subjects: 61.3 +/- 20.2 units/mg protein (mean +/- SD; n = 50) versus 46.6 +/- 22.2 units/mg protein (n = 49; p = 0.0002). There was a significant correlation of hepatic catechol-O-methyltransferase activity and thermal stability in samples from both female (rs = 0.698; p = 0.0001) and male subjects (rs = 0.429; p = 0.002). Finally, when both red blood cell catechol-O-methyltransferase activity and thermal stability were measured in blood samples from 34 of these patients, there was a significant correlation between catechol-O-methyltransferase heated/control ratios and levels of enzyme activity in hepatic tissue and in red blood cell lysates. These findings indicate that the genetic polymorphism that controls catechol-O-methyltransferase activity level and thermal stability in red blood cells also controls those same properties of the enzyme in the human liver.

N-Acetyl-6-hydroxytryptophan oxidase, a developmentally controlled phenol oxidase from Aspergillus nidulans

We have purified a specific phenol oxidase which is produced during conidiophore development in the fungus Aspergillus nidulans. Two active forms (A and B) have molecular masses of 50 and 48 kDa respectively; they have identical N-termini (24 residues). We have analysed the metal ion content of the B form; it is unusual in consisting of one zinc and two copper atoms per molecule. A temperature-sensitive mutant (ivoB192) produces a thermolabile enzyme, implying that ivoB is the structural locus. The natural substrate of the enzyme is N-acetyl-6-hydroxytryptophan, but it can be assayed colorimetrically or polarographically using hydroquinone monomethyl ether (HME) as substrate. It will also oxidize p-cresol, but not tyrosine, 3,4-dihydroxyphenylalanine or o-methoxyphenol. Colour development with HME substrate is strongly enhanced by high ammonium ion concentrations. Activity against HME is inhibited by 2,3-dihydroxynaphthalene, phenylhydrazine, diethyl dithiocarbamate and 8-hydroxyquinolene.

Human Aldolase A of a Hemolytic Anemia Patient with Asp-128→Substitution: Characteristics of an Enzyme Generated in E. coli Transfected with the Expression Plasmid pHAAD128G1

Aldolase A derived from a hemolytic anemia patient with aldolase A deficiency was shown to have an amino acid substitution of glycine for aspartic acid at the 128th position (Asp-128) in the enzyme [Kishi et al. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8623-8627]. We constructed an Escherichia coli expression plasmid, pHAAD128G, which carries the mutant aldolase A [aldolase A(D-G)] cDNA, and the enzyme generated in E. coli transfected with the expression plasmid was purified and characterized. Conversion of Asp to Gly at the 128th position in the enzyme rendered the enzyme thermolabile and susceptible to tryptic digestion. CD spectra analysis also revealed that the mutant enzyme had a remarkable conformation change with a decrease of regular form in the molecule. Addition of glycerol or some other polyalcohols during thermal treatment protected this altered enzyme (but not the normal enzyme) against denaturation and activity decrease. In order to determine the function of the amino acid residue at the 128th position, two artificial mutant enzymes with the substitutions of Glu for Asp [aldolase A(D-E)] and Ser for Asp [aldolase A(D-S)], respectively, at the position were constructed by site-directed mutagenesis and characterized. These analyses demonstrated the necessity for Asp to be present at the 128th residue in order for this enzyme to be thermally stable.

Characteristics of thermostable pullulanase from Bacillus stearothermophilus and the nucleotide sequence of the gene

Thermostable pullulanase was purified to homogeneity on sodium dodecyl sulfate-polyacrylamide gel from the culture supernatant of Bacillus stearothermophilus TRS128. However, multiformity of the pullulanase was suggested by activity staining on a pullulan-reactive red plate. The thermostability of the enzyme was tested. In the presence of Ca 2+, the optimum temperature of the pullulanase was 75°C, and nearly 100% of the enzyme activity was retained even after treatment at 68°C for 60 min. Since the thermostable pullulanase gene ( pulT) has been cloned, the nucleotide sequence was determined. Although the DNA sequence revealed only one large open reading frame, two possible pairs of SD sequence and initiation codon were found in the frame. To analyze the regulatory region, several mutations (deletion, insertion and substitution of nucleotides) were introduced in the flanking region of pulT, using site-directed mutagenesis. A putative promoter, SD sequence and initiation codon were inferred. The pulT gene was composed of 1974 bases and 658 amino acid residues (molecular weight 75,375). The deduced amino acid sequence of the thermostable pullulanase exhibited a fairly low homology with that of the thermolabile pullulanase from Klebsiella aerogenes. However, four consensus sequences containing catalytic and/or substrate binding sites for amylolytic enzymes were also found in the thermostable pullulanase and the thermolabile enzyme.

Identification of essential histidine residues in the active site of Escherichia coli xylose (glucose) isomerase.

Two conserved histidine residues (His-101 and His-271) appear to be essential components in the active site of the enzyme xylose (glucose) isomerase (EC 5.3.1.5). These amino acid residues were targeted for mutagenesis on the basis of sequence homology among xylose isomerases isolated from Escherichia coli, Bacillus subtilis, Ampullariella sp. strain 3876, and Streptomyces violaceus-niger. Each residue was selectively replaced by site-directed mutagenesis and shown to be essential for activity. No measurable activity was observed for any mutations replacing either His-101 or His-271. Circular dichroism measurements revealed no significant change in the overall conformation of the mutant enzymes, and all formed dimers similar to the wild-type enzyme. Mutations at His-271 could be distinguished from those at His-101, since the former resulted in a thermolabile protein whereas no significant change in heat stability was observed for the latter. Based upon these results and structural data recently reported, we speculate that His-101 is the catalytic base mediating the reaction. Replacement of His-271 may render the enzyme thermolabile, since this residue appears to be a ligand for one of the metal ions in the active site of the enzyme.

Monitoring the Degradation of Commercial Microbial Rennet Preparations

Fractionation on a DEAE-Sephadex A-50 column was used to study the degradation of microbial rennets derived from Mucor miehei. A comparison was made between the crude culture filtrate, derived from the growth of the microorganism in submerged culture condition on wheat bran medium, and commercial preparation Marzyme and Rennilase. The elution protein profiles lacked some of the peaks associated with the crude culture filtrate however, the milk-coagulating activity eluted out at approximately the same NaCl molarity (.45 M). Each brand name product had a characteristic elution pattern, which did not vary between their thermostable and thermolabile enzyme preparations. With storage at 4°C the elution protein profiles of the thermolabile enzyme preparations changed in a distinctive manner characteristic of the brand name. With the Marzyme series, the milk-coagulating activity eluted out at the same NaCl molarity, whereas other established peaks increased in area. The milk-coagulating activity, from Rennilase, eluted from the column at a lower NaCl concentration (.35 M). The changes were evidence of enzyme degradation, which was manifested by a general decrease in specific enzyme activity. By using column chromatography it was possible to monitor the stages of degradation with time.

Effect of procaine on cultivated human WI-38 fibroblasts

Procaine is a local anesthetic, also used in experimental gerontology and has been tested in cultivated human WI-38 fibroblasts. This molecule was found to enhance growth rate and cell densities in actively dividing cultures. As the cells aged, however, this stimulatory effect diminished and finally vanished. In a long term experiment the enhancement of growth of procaine treated cultures was finally replaced by a toxic effect even at low concentration. The amount of the thermolabile enzyme found in phase III cells did not change when procaine was added to the culture medium. In this cellular aging model, procaine behaved like a metabolic stimulator of actively dividing cells but not as an “antiaging” molecule as it is sometimes assumed.

Genetic analysis of the gene ICL1 of the yeast Yarrowia lipolytica.

The gene ICL1 codes for the tetrameric enzyme isocitrate lyase of Y. lipolytica. Twenty icl1- alleles have been analysed for their reversion frequency, their interallelic complementation pattern, and the position of the corresponding mutation site on the fine structure map of the gene ICL1. One intragenic temperature-sensitive revertant of the allele icl1D-39 was isolated, which expressed a thermolabile enzyme. In spite of the fact that nonsense mutations have been detected, the direction of transcription of the gene ICL1 was inferred from the localization of a linked cis-dominant regulatory mutation site. The size of the mitotic map of this gene suggests that recombination frequency in Y. lipolytica is lower than in Saccharomyces cerevisiae.

Human triose-phosphate isomerase deficiency: a single amino acid substitution results in a thermolabile enzyme.

Triose-phosphate isomerase (TPI; D-glyceraldehyde-3-phosphate ketol-isomerase, EC 5.3.1.1) deficiency is a recessive disorder that results in hemolytic anemia and neuromuscular dysfunction. To determine the molecular basis of this disorder, a TPI allele from two unrelated patients homozygous for TPI deficiency was compared with an allele from a normal individual. Each disease-associated sequence harbors a G X C----C X G transversion in the codon for amino acid-104 and specifies a structurally altered protein in which a glutamate residue is replaced by an aspartate residue. The importance of glutamate-104 to enzyme structure and function is implicated by its conservation in the TPI protein of all species that have been characterized to date. The glutamate-to-aspartate substitution results in a thermolabile enzyme as demonstrated by assays of TPI activity in cultured fibroblasts of each patient and cultured Chinese hamster ovary (CHO) cells that were stably transformed with the mutant alleles. Although this substitution conserves the overall charge of amino acid-104, the x-ray crystal structure of chicken TPI indicates that the loss of a side-chain methylene group (-CH2CH2COO- ---- -CH2COO-) is sufficient to disrupt the counterbalancing of charges that normally exists within a hydrophobic pocket of the native enzyme.

Immunochemical differences among molecular forms of acetylcholinesterase in brain and blood.

Molecular forms of acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) differ in their solubility properties as well as in the number of their catalytic subunits. We used monoclonal antibodies to investigate the structure of acetylcholinesterase forms in brain, erythrocytes and serum of rats, rabbits and other mammals. Two antibodies were found to bind tetrameric acetylcholinesterase in preference to the monomeric enzyme. These antibodies also displayed lower affinity for certain forms of 'soluble' brain acetylcholinesterase than for the 'membrane-associated' counterparts. Furthermore, one of them was virtually lacking in affinity for the membrane-associated enzyme of erythrocytes. The basis for the antibody specificity was not fully determined. However, the immunochemical results were supported by measurements of enzyme thermolability, which showed that the catalytic activity of 'soluble' acetylcholinesterase was comparatively heat-resistant. These observations point toward structural differences among the solubility classes of acetylcholinesterase.

Aspartate metabolism in Lactobacillus murinus CNRS 313. I. Aspartase.

Aspartase from Lactobacillus murinus CNRZ 313 (L-aspartate ammonia lyase, E.C. 4.3.1.1.), is one of the enzymes of aspartate metabolism in lactic acid bacteria. The growth of this microorganism in LAPTg medium is not altered by the addition of L-aspartate. Aspartase is a thermolabile enzyme. Its activity was not changed in one month at temperatures lower than 0°C, independently of the presence of 10% glycerol or 0.05M MgCl2. Maximum activity occurred at 32°C and pH 7.5 in 0.15M Tris-HCl pH 7.5. At pH values different from the optimum, positive cooperation between substrate molecules was observed.The Km values for L-aspartate at pH 7.5 and 7.0 were: 3.7×10-2M and 9.0×10-2M respectively. The pK values of pKa 6.75 and pKb 7.35 were calculated from Dixon plots. The ΔG of the reaction was calculated by Arrhenius plot. The values were 4, 807cal mol-1 below 25°C and 2, 665cal mol-1 above 25°C. Both 10-3M HgCl2 and NaCN had inhibitory effects.

8] Glutaminyl-tRNA synthetase of Escherichia coli

Each amino acid to be incorporated into proteins by mRNA directed protein synthesis is activated by a specific aminoacyl-tRNA synthetase. Bacteria have only one synthetase specific for each amino acid, and this enzyme recognizes all tRNA species (isoacceptors) for the cognate amino acid. Glutamine and glutamate are activated by separate enzymes in E. coli . In contrast, gram-negative bacteria form Gln-tRNA Gln in a multistep process starting with the synthesis by glutamyl-tRNA synthetase (GluRS) of Glu-tRNA Gln and its subsequent transformation by an amidotransferase to Gln-tRNA Gln . The structural gene for glutaminyl-tRNA synthetase (GlnRS), glnS , is located near min 14 on the E. coli chromosome. Mutants of this synthetase give rise to a thermolabile enzyme or to a protein with altered tRNA recognition properties (mischarging). The cellular level of GlnRS is regulated both metabolically and by a gene unlinked to glnS . The structural gene for GlnRS is isolated on a 6.5-kb DNA fragment and incorporated into pBR322 to form the plasmid pYY105. This plasmid can rescue the temperature-sensitive phenotype of the glnS mutation.

Determination of methylumbelliferyl-N-acetylneuraminic acid sialidase for clinical purposes.

In order to determine optimal conditions for the clinical application of leukocyte acid sialidase determination, we have studied some characteristics of the enzyme giving attention more specifically to the enzyme's thermolability and the influence of potential effectors. The enzyme has a pH optimum around 4 and a Km value of 0.25 mmol . 1(-1). A decrease of activity is observed when the enzyme is measured in leukocytes isolated from total blood or leukocytes containing plasma kept for several hours at room temperature. On the other hand, in a leukocyte homogenate, the enzyme is stable at 0 degree C for 8 hours although pronounced thermolability is observed at 23 and 37 degrees C and total inactivation occurs after 10 minutes at 50 degrees C. The addition of albumin to the homogenate partially protects the enzyme. Residual activities of 40% and 50% were measured in leukocytes and leukocyte homogenates frozen during 2 months at -20 degrees C. A marked inhibition was obtained by Triton X-100, sodium taurocholate, and methoxyphenyl-N-acetylneuraminic acid. Immediate isolation of the leukocytes after the blood drawing and subsequent homogenization at 0 degree C of a dense pellet make it possible to achieve enzyme linearity and reproducible results.

Hepatic microsomal NADPH-cytochrome P-450 reductase from little skate, Raja erinacea. Comparison of thermolability and other molecular properties with a mammalian enzyme

Components of little skate (an elasmobranch) and rabbit hepatic microsomal cytochrome P-450 dependent monooxygenase systems were examined for differences which might explain the decreasing xenobiotic-metabolizing activity of little skate microsomes assayed at temperatures above 30°C. The proportion of saturated fatty acids in microsomal lipids and the habitat temperature are both lower in skate as compared to rabbit, which is consistent with the known adaptive pattern. The more thermolabile enzyme of the skate system in microsomal preparations is NADPH-cytochrome P-450 reductase. The optimal assay temperature for purified skate reductase (30°C) is 10°C lower than that for the purified rabbit reductase. The purified skate reductase differs from rabbit reductase in monomeric molecular weight, in peptides produced by partial proteolysis, in immunochemical properties, but not in flavin content.

Molybdenum cofactor requirement for biotin sulfoxide reduction in Escherichia coli.

The bisC gene of Escherichia coli is tentatively identified as the structural gene for biotin sulfoxide reductase by the isolation of bisC(Ts) mutants that make thermolabile enzyme. The products of four other E. coli genes (chlA, chlB, chlE and chlG) are also needed for enzymatic activity. Mutations previously assigned to the bisA, bisB, and bisD genes belong to genes chlA, chlE, and chlG, respectively. The biotin sulfoxide reductase deficiency of a chlG, mutant is partially reversed by the addition of 10 mM molybdate to the growth medium. Mutational inactivation of the chlD gene reduces the specific activity of biotin sulfoxide reductase about twofold. This effect is reversed by the addition of 1 mM molybdate to the growth medium. The specific activity of biotin sulfoxide reductase is decreased about 30-fold by the presence of tungstate in the growth medium, an effect that has been observed previously with nitrate reductase and other molybdoenzymes. The specific activity of biotin sulfoxide reductase is not elevated in a lysate prepared by derepressing a lambda cI857 chlG prophage. Whereas biotin sulfoxide reductase prepared by sonic extraction of growing cells is almost completely dependent on the presence of a small heat-stable protein resembling thioredoxin, much of the enzyme obtained from lysates of thermoinduced lambda cI857 lysogens does not require this factor.