Optimum pH For Activity Research Articles

Murine Liver Homogentisate 1,2-Dioxygenase. Purification to Homogeneity and Novel Biochemical Properties

1. The murine liver enzyme homogenisate 1,2-dioxygenase (HGO) is purified 330-fold. The molecular mass, as determined by gel filtration, is approximately 149 kDa. SDS/PAGE under strongly reducing conditions reveals a single band at 49 kDa, whose concentration increases during the purification. 2. HGO has a pI = 8. The pH optimum for activity in vitro is 6.1. 3. The Km for its natural substrate HGA is 188 microM, whereas the Kd for the ferrous ion is determined at 19 microM. Fe2+ is an absolutely obligate cofactor and cannot be replaced by other divalent cations. Incubating the enzyme with Fe2+ plus one of other divalent cations Zn2+, Cu2+, Co2+ or Ni2+ in equimolar concentrations inhibits HGO, whereas addition of Ca2+ and Mn2+ has no effect. Ascorbate probably acts as a reducing agent, protecting Fe2+ from spontaneous oxidation or by reversing its oxidation. Ascorbate has a great potential to stabilize the assay system. 4. The activation energy determined by an Arrhenius plot is 24 kJ/mol. 5. HGO consists of a single type of subunit with no intermolecular disulfide bridges and requires Fe2+ as a cofactor. This emphazises that the enzyme is familiar with the class of extradiol dioxygenases.

Murine Liver Homogentisate 1,2-Dioxygenase. Purification to Homogeneity and Novel Biochemical Properties

1. The murine liver enzyme homogenisate 1,2-dioxygenase (HGO) is purified 330-fold. The molecular mass, as determined by gel filtration, is approximately 149 kDa. SDS/PAGE under strongly reducing conditions reveals a single band at 49 kDa, whose concentration increases during the purification. 2. HGO has a pI = 8. The pH optimum for activity in vitro is 6.1. 3. The Km for its natural substrate HGA is 188 microM, whereas the Kd for the ferrous ion is determined at 19 microM. Fe2+ is an absolutely obligate cofactor and cannot be replaced by other divalent cations. Incubating the enzyme with Fe2+ plus one of other divalent cations Zn2+, Cu2+, Co2+ or Ni2+ in equimolar concentrations inhibits HGO, whereas addition of Ca2+ and Mn2+ has no effect. Ascorbate probably acts as a reducing agent, protecting Fe2+ from spontaneous oxidation or by reversing its oxidation. Ascorbate has a great potential to stabilize the assay system. 4. The activation energy determined by an Arrhenius plot is 24 kJ/mol. 5. HGO consists of a single type of subunit with no intermolecular disulfide bridges and requires Fe2+ as a cofactor. This emphazises that the enzyme is familiar with the class of extradiol dioxygenases.

Murine Liver Homogentisate 1,2-Dioxygenase. Purification to Homogeneity and Novel Biochemical Properties

1 The murine liver enzyme homogenisate 1,2-dioxygenase (HGO) is purified 330–fold. The molecular mass, as determined by gel filtration, is approximately 149 kDa. SDS/PAGE under strongly reducing conditions reveals a single band at 49 kDa, whose concentration increases during the purification. 2 HGO has a pI = 8. The pH optimum for activity in vitro is 6.1. 3 The Km for its natural substrate HGA is 188 μM, whereas the Kd for the ferrous ion is determined at 19 μM. Fe2+ is an absolutely obligate cofactor and cannot be replaced by other divalent cations. Incubating the enzyme with Fe2+ plus one of other divalent cations Zn2+, Cu2+, Co2+ or Ni2+ in equimolar concentrations inhibits HGO, whereas addition of Ca2+ and Mn2+ has no effect. Ascorbate probably acts as a reducing agent, protecting Fe2+ from spontaneous oxidation or by reversing its oxidation. Ascorbate has a great potential to stabilize the assay system. 4 The activation energy determined by an Arrhenius plot is 24 kJ/mol. 5 HGO consists of a single type of subunit with no intermolecular disulfide bridges and requires Fe2+ as a cofactor. This emphazises that the enzyme is familiar with the class of extradiol dioxygenases.

The NADPH oxidase of phagocytic cells is an electron pump that alkalinises the phagocytic vacuole

Phagocytic cells of the immune system contain an oxidase that is important for the killing and digestion of engulfed microbes. This is an electron transport chain that transfers electrons from NADPH in the cytosol to oxygen to form superoxide and hydrogen peroxide in the phagocytic vacuole. Absence or abnormality of this oxidase results in the syndrome of CGD, characterised by a profound predisposition to infection. The electron transport chain consists of a flavocytochrome b located in the plasma membrane and membrane of the specific granules. It is composed of a and b-subunits, with apparent molecular masses of 23 kDa and 76–92 kDa, respectively. The b-subunit is a member of the FNR family of reductases with FAD and NADPH binding sites. Based upon the crystal structure of FNR we have constructed a model of the more hydrophilic C terminal half of this b-subunit, which acts as a guide to the organisation of the molecule, and provides a template on which to map mutations in CGD. The location of the heme is uncertain. Electron transport is dependent upon an activation complex of cytosolic proteins including p40phox, p47phox, and p67phox, and the small GTP binding protein, p21rac. This oxidase system is important for the killing and digestion of bacteria and fungi. This might be accomplished in a number of ways. The oxidase produces superoxide and hydrogen which might be toxic themselves. The hydrogen peroxide can act as substrate for myeloperoxidase which can oxidise chloride and iodide to chlorine and iodine and their hypohalous acids. The proteins contained within the cytoplasmic granules are also very important in the killing process. These are neutral proteinases that require a neutral or slightly alkaline pH for optimal activity. The oxidase transports electrons, unaccompanied by protons, across the wall of the phagocytic vacuole, resulting in an elevation of the vacuolar pH, thereby optimising conditions for killing and digestion of engulfed organisms by these neutral proteinases.

FURTHER CHARACTERIZATION OF THE 12 kDa PROTEASE/ALPHA AMYLASE INHIBITOR PRESENT IN MAIZE SEEDS

A 12 kDa protease/α-amylase inhibitor was purified from maize seeds and studied. Its trypsin- and amylase-inhibitor activities against enzymes from different origins were determined, as well as its optimal pH for inhibition. Eight different proteases, extracted from insects and fungi which attack grains during storage, were tested with the inhibitor. Bovine trypsin and trypsin-like proteases from the insect P. truncatus, and the fungi A. niger and A. fumigatus, were recognized by this inhibitor. Out of 11 α-amylases tested, only those from T. castaneum and C. maculatus were recognized by this inhibitor. The optimal pH's for the inhibition of trypsin and the trypsin-like protease from P. truncatus were 8.0 and 7.5, respectively. The optimal pH activity was 5.0 for the inhibition of amylases from T. castaneum and C. maculatus.

Purification and characterization of a protease-resistant cellulase from Aspergillus niger

An endo-β-1,4-glucanase (EC 3.2.1.4) was purified from a culture filtrate of Aspergillus niger IFO31125 by column chromatography through TSK-gel DEAE-3SW and TSK-gel DEAE-5PW, and by gel filtration through TSK-gel G2000SW by high performance liquid chromatography. The enzyme was estimated to have a molecular weight of about 40 kDa by both gel filtration and SDS-polyacrylamide gel electrophoresis, and appeared to consist of a monomeric protein. It contained 8.9% carbohydrate. The optimal pH for activity was 6.0–7.0, and the stable pH range was 5.0–10.0. The optimum temperature at pH 6.0 was around 70°C. The enzyme was very thermally stable and no loss of original activity was found on incubation at 60°C for 2 h. The enzyme efficiently hydrolyzed carboxymethylcellulose and lichenan, but crystalline forms of cellulose, curdlan, laminarin, cellobiose, p-nitrophenyl-β- d-glucopyranoside and p-nitrophenyl-β- d-cellobioside were barely hydrolyzed. The activity of the enzyme was inhibited by Hg 2+ and Cu 2+ but was not affected by other inhibitors of thiol enzymes such as p-chloromercuribenzoate and N-ethylmaleimide. N-Bromosuccinimide showed a strong inhibitory effect, suggesting that a tryptophan residue is essential for the activity of the enzyme. The N-terminal amino acid sequence of the enzyme showed considerable homology to those of endo-β-1,4-glucanases from some other microorganisms, including Sclerotinia sclerotiorum and Schizophyllum commune. The enzyme had very strong protease-resistance, and showed no loss of activity when incubated with proteases such as Savinase at 40°C, even for 2 weeks.

A serine proteinase of a fungus isolated from dried bonito “Katsuobushi”

A proteinase was purified from an extract of the koji culture of a strain of Aspergillus sydowi, isolated from “Katsuobushi”, in a homogeneous state by polyacrylamide gel electrophoresis by ammonium sulfate fractionation, and Butyl-Toyopearl, DEAE-Toyopearl and Bio-Gel P-100 column chromatographies. The molecular weight to the enzyme was calculated to be about 40,000. The optimum pH activity was 7.0–7.5 at 37°C and the optimum temperature was 45°C at pH 7.2 for caseinolytic activity. The enzyme was stable between pH 3.5–8.0 and a temperature of up to 45°C. As the enzyme activity was completely inhibited with phenylmethylsulfonylfluoride, this enzyme was a kind of serine proteinase. The enzyme had a different substrate specificity from other fungal serine proteinases reported so far from the results of cleavage of proangiotensin and oxidized insulin B-chain.

Homogeneous pyrimidine nucleotidase from human erythrocytes: enzymic and molecular properties

A pyrimidine nucleotidase with unique specificity has been obtained for the first time as an homogeneous protein from the cytosolic fraction of human erythrocytes. Both conventional chromatography and f.p.l.c. techniques have been used in the purification procedure. The final enzyme preparation gave a single protein band of M(r) = 23,500 on SDS/PAGE under both reducing and non-reducing conditions. The native enzyme was eluted at M(r) = 45,000 in gel filtration chromatography on Superose 12, suggesting a dimeric structure. Amino acid analysis was consistent with an acidic isoelectric point and revealed the presence of six half-cystine and two methionine residues per subunit. The enzyme was active on a variety of pyrimidine nucleoside monophosphates, being most active on the 3'-monophosphates. Km values for 3'dUMP, 3'UMP, 5'dUMP, 5'UMP, 5-fluoro-2'dUMP, ranged from 192 microM to 1.15 mM. The enzyme activity was inhibited by both reaction products, orthophosphate, and the nucleoside formed. Product inhibition studies suggested an Ordered Uni-Bi mechanism for the reaction. The enzyme required Mg2+ for its activity, while heavy metals cations were strongly inhibitory. The enzyme activity was inhibited by metal chelating agents and it was sensitive to thioreactive reagents. The isoelectric point was 5.4 and the optimum activity pH was 6.5.

Dienelactone hydrolase from Rhodococcus erythropolis 1 CP: purification and properties

Dienelactone hydrolases (EC 3.1.1.45) have been shown to play an indispensable role in the degradation of chloroaromatic compounds via ortho-cleavage of chlorocatechols. We report on the purification of dienelactone hydrolase of the chlorophenol-utilizing strain Rhodococcus erythropolis 1CP to apparent homogeneity. Dienelactone hydrolase differed fron the corresponding enzymes of other chloroaromatic compound-catabolizing strains in being restricted to substrates with a cis-dienelactone structure. From the cis-dienelactone-hydrolyzing enzyme of a 4-fluorobenzoate-utilizing Burkholderia (Pseudomonas) cepacia strain, it differed considerably in properties such as pH optimum of activity, inhibition by p-chloromercuribenzoate, and amino acid composition. Thus, there is not necessarily a close relationship between substrate specificity and other properties of dienelactone hydrolases.

Enzymatic Activity of Desquamin

We have previously described the properties of desquamin, a cell adhesion molecule in the stratum corneum with lectin-like properties specific for amine sugars. We report here that desquamin is also a trypsin-like serine proteinase. It degrades several chromogenic peptides with arginine in the P1 position, with greatest activity for the tissue plasminogen activator peptide; it has no chymotrypsin-like activity. The enzymatic activity of desquamin is inhibited by aprotinin, leupeptin, and soybean trypsin inhibitor. The Km for all active substrates is in the millimole range and the pH for optimal activity is near 10. The enzymatic activity is stable in the temperature range from 37 to 80°C, peaking near the upper end; it is only partially inhibited at 100°C. Using zymogels with immobilized substrates, we show that desquamin degrades both casein and human keratins. Because desquamin is localized to the lipid envelopes of the stratum corneum and can function as an enzyme (and is extremely resistant to chemical and thermal degradation), it is in a position to play a crucial role in desquamation.

A new blood-coagulating protease in mitochondrial membranes of rat submaxillary glands. Purification and characterization of protease and its blood-coagulating activity.

An integral membrane protease was solubilized and purified to homogeneity from rat submaxillary mitochondria. The purified enzyme could coagulate rabbit plasma. The molecular mass of the enzyme is 22 kDa on SDS-polyacrylamide gel electrophoresis under reducing conditions and 24 kDa on gel filtration on a Sephadex G-100 column. Its isoelectric point is 4.2-4.25. Enzyme activity is strongly inhibited by diisopropyl fluorophosphate, soybean trypsin inhibitor, benzamidine, aprotinin, and antipain, suggesting the enzyme as a serine protease. Its pH optimum for activity is 8.5. Zn2+ is strongly inhibitory; at 1 mM concentration it produced 72% inhibition. The enzyme is active toward different synthetic substrates (p-nitroanilide derivatives) containing Arg at the P1 position with blocked NH2 terminus. Kcat/Km was highest with the substrate N-Bz-Pro-Arg-pNa (where Bz is benzoyl and pNA is paranitroanilide). The purified enzyme coagulates rabbit plasma in a dose-dependent manner. Plasma coagulation by the enzyme is completely blocked in the presence of aprotinin or soybean trypsin inhibitor, suggesting that protease activity is required for this coagulation reaction. Antibody raised against the purified enzyme inhibits the plasma coagulation initiated by the enzyme. The enzyme can correct the prolonged clotting time of factor X-deficient human plasma but is unable to convert purified fibrinogen to fibrin clots, indicating factor Xa-like activity of the enzyme. The enzyme has the ability to activate prothrombin. Several properties of the enzyme distinguish it from other reported submaxillary proteases.

Purification and Characterization of 3-Methylcrotonyl-Coenzyme-A Carboxylase from Leaves of Zea mays

3-Methylcrotonyl-CoA carboxylase has been purified to near homogeneity from maize leaves. The resulting preparations of 3-methylcrotonyl-CoA carboxylase have a specific activity of between 200 and 600 nmol · min−1 · mg−1 protein, representing an approximately 5000-fold purification of the enzyme. The purified 3-methylcrotonyl-CoA carboxylase has a molecular weight of 853,000 ± 34,000 and is composed of two types of subunits, a biotin-containing subunit of 80 ± 2 kDa and a non-biotin-containing subunit of 58.5 ± 1.5 kDa. These data suggest that the enzyme has an α6β6 configuration. The optimum pH for activity is 8.0. The kinetic constants for the substrates 3-methylcrotonyl-CoA, ATP, and HCO−3 are 11 μM, 20 μM, and 0.8 mM, respectively. Kinetic studies of the 3-methylcrotonyl-CoA carboxylase reaction with variable concentrations of two substrates confirmed that ATP and HCO−3 bind sequentially to the enzyme and that ATP and 3-methylcrotonyl-CoA bind in ping-pong fashion. However, similar analyses indicate that the binding of HCO−3 at the first site is affected by 3-methylcrotonyl-CoA. Kinetic studies of the role of Mg2+ in the 3-methylcrotonyl-CoA carboxylase reaction establish that Mg · ATP is the substrate for the enzyme, that free ATP is an inhibitor, and that free Mg2+ is an activator. Both Mn2+ and Co2+ can substitute somewhat for Mg2+, but Zn2+ is unable to do so. In addition to carboxylating 3-methylerotonyl-CoA, the maize carboxylase can carboxylate crotonyl-CoA, but not acetoacetyl-CoA. In fact, acetoacetyl-CoA is a potent, noncompetitive inhibitor, which indicates that the enzyme contains an acetoacetyl-CoA binding site that is independent of the active sites. The monovalent cations K+, Cs+, Rb+, and NH+4 activated 3-methylcrotonyl-CoA carboxylase activity, with Rb+ being the most potent activator. The inhibition of 3-methylcrotonyl-CoA carboxylase by sulfhydryl and arginyl modifying reagents could be partly alleviated by the substrates ATP and 3-methylcrotonyl-CoA, which suggests that sulfhydryl and arginyl residues may be involved in catalysis.

Characterization of a new Bacillus stearothermophilus isolate: a highly thermostable α-amylase-producing strain

A novel strain of Bacillus stearothermophilus was isolated from samples of a potato-processing industry. Compared to known α-amylases from other B. stearothermophilus strains, the isolate was found to produce a highly thermostable α-amylase. The half-time of inactivation of this α-amylase was 5.1 h at 80°C and 2.4 h at 90°C. The temperature optimum for activity of the α-amylase was 70°C; the pH optimum for activity was relatively low, in the range 5.5–6.0. α-Amylase synthesis was regulated by induction and repression mechanisms. An inverse relationship was found between growth rate and α-amylase production. Low starch concentrations and low growth temperatures were favourable for enzyme production by the organism. At the optimal temperature for growth, 65°C, the α-amylase was a growth-associated enzyme. The optimal temperature for α-amylase production, however, was 40°C, with α-amylase increasing from 3.9 units (U)/ml to 143 U/ml when lowering the growth temperature from 65°C to 40°C. Maximal α-amylase production in a batch fermentor run at 65°C was 102 U/ml, which was 26-fold higher than in erlenmeyer flasks at 65°C. The dissolved O2 concentration was found to be a critical factor in production of the α-amylase.

Characterization of a new Bacillus stearothermophilus isolate: a highly thermostable ?-amylase-producing strain

A novel strain of Bacillus stearothermophilus was isolated from samples of a potato-processing industry. Compared to known α-amylases from other B. stearothermophilus strains, the isolate was found to produce a highly thermostable α-amylase. The half-time of inactivation of this α-amylase was 5.1 h at 80°C and 2.4 h at 90°C. The temperature optimum for activity of the α-amylase was 70°C; the pH optimum for activity was relatively low, in the range 5.5–6.0. α-Amylase synthesis was regulated by induction and repression mechanisms. An inverse relationship was found between growth rate and α-amylase production. Low starch concentrations and low growth temperatures were favourable for enzyme production by the organism. At the optimal temperature for growth, 65°C, the α-amylase was a growth-associated enzyme. The optimal temperature for α-amylase production, however, was 40°C, with α-amylase increasing from 3.9 units (U)/ml to 143 U/ml when lowering the growth temperature from 65°C to 40°C. Maximal α-amylase production in a batch fermentor run at 65°C was 102 U/ml, which was 26-fold higher than in erlenmeyer flasks at 65°C. The dissolved O2 concentration was found to be a critical factor in production of the α-amylase.

Increased thermostability of Asn182 → Ala mutant Aspergillus awamori glucoamylase

Asn182 --> Ala Aspergillus awamori glucoamylase expressed in Saccharomyces cerevisiae had a first-order thermodeactivation coefficient 40% that of wild-type glucoamylase at pH 4.5 between 60 degrees and 65 degrees C, caused by the elimination of an Asn-Gly sequence subject to deamidation and eventual chain breakage. Above 70 degrees C, and at pHs 3.5 and 5.5, thermodeactivation coefficients of wild-type and mutant enzymes were roughly equal, because the fastest deactivation mechanism was no longer deamidation. The mutation had little effect on the enzyme's optimal pH for activity and subsite map, or on the glucose yield from starch dextrin hydrolysis. During enzyme production by yeast fermentation, highest cell densities and activities of wild-type and mutant glucoamylases were attained after a period of glucose starvation, followed by a second addition of glucose. (c) 1994 John Wiley & Sons, Inc.

Purification of pectate lyase produced by Colletotrichum gloeosporioides and its inhibition by epicatechin: a possible factor involved in the resistance of unripe avocado fruits to anthracnose.

Pectate lyase (PL) from Colletotrichum gloeosporioides was purified to apparent homogeneity by hydrophobic interaction chromatography followed by isoelectric focusing. The purified preparation showed one band corresponding to 40 kD on sodium dodecyl sulfate-polyacrylamide gels. The isoelectric point of the enzyme was 7.9, and the optimum pH for activity was 8.9. The purified PL efficiently macerated unripe avocado fruit wedges. In vitro translation of mRNA from an induced fungal culture revealed a 36-kD precursor polypeptide, which was precipitated with PL antibodies. The antibodies inhibited enzymatic activity and maceration ability on avocado wedges. Epicatechin, a flavan 3-ol present in the peel of unripe avocado fruit, had a Ki of 3.4 microM for inhibition of PL activity in vitro. At 20 micrograms/ml (68 microM), epicatechin reduced the enzyme's macerating ability by 64%. Since the flavan is present in unripe fruit at much higher concentrations (about 350 micrograms/g fresh weight) than the inhibitory concentrations, epicatechin may be involved in the resistance of unripe avocado fruits by inhibiting the PL activity of C. gloeosporioides.

Secretion, purification and characterization of a soluble form of the yeast KEX1-encoded protein from insect-cell cultures.

The Saccharomyces cerevisiae KEX1 gene encodes a carboxypeptidase involved in the C-terminal processing of the lysine and arginine residues from the precursors of K1 and K2 killer toxins and alpha-factor (mating pheromone). In order to produce large quantities of this unique carboxypeptidase for structural studies, a functional soluble form was obtained by deleting 224 amino acids from the C-terminus of the KEX1-encoded protein which includes a putative membrane-spanning domain. The resulting truncated KEX1 gene (KEX1 delta) has been expressed in the baculovirus/insect cell system. The protein (Kex1 delta p) is efficiently secreted into the culture medium and was purified to apparent homogeneity with a yield of approximately 4 mg/l culture. Kex1 delta p is a glycoprotein with a molecular mass of 56 kDa, its N-terminal sequence is identical to that of the full-length membrane-associated form of the enzyme [Latchinian-Sadek, L. & Thomas, D. Y. (1993) J. Biol. Chem. 268, 534-540], and like the full-length enzyme it is not made as a proenzyme. For the soluble enzyme form, the optimum pH for activity was 5.5-6.0, and the apparent pI value of the protein determined by isoelectric focusing was 4.2. The enzyme cleaves arginine from the C-terminus of the synthetic peptide benzoyl-Phe-Ala-Arg with Km 335 microM and Vmax 282 mumol.min-1 x mg protein-1. Insect-cell-derived Kex1 delta p processes alpha-factor-Lys-Arg, a known natural substrate, to mature active alpha-factor in a manner similar to the membrane-associated full-length enzyme. This secreted form of the enzyme is a convenient source for the isolation of substantial quantities of the pure enzyme for detailed kinetic and structural studies.

The Enzymatic Production of Adipic Acid

Mutants of Brevibacterium sp. R312 were isolated for the production of adipic acid from 1,4-dicyanobutane (adiponitrile). One mutant (Ad), with a modified cell wall showed activity against adipamide three times greater than the wild type. Another mutant (ACV2) derived from the Ad strain had 30 times more activity on 5-cyanovaleric acid, and 7 times more on adipamide than the wild type.The nitrile hydratase from the mutant strain ACV2 was purified and compared to that from the wild type R312. The nitrile hydratase of the mutant strain is different from that of the wild type by its pHi, optimum activity pH, and its rates of hydrolysis of 5-cyanovaleramide and 5-cyanovaleric acid which were 30 and 15 folds greater.The presence of a new amidase named “adipamidase” acting on amide intermediates in the hydrolysis of dinitriles to organic acids was demonstrated in this mutant ACV2.

Characterization of an SH2 containing protein tyrosine phosphatase in rat parotid gland acinar cells

Rat parotid glands were shown to possess protein phosphatase activity capable of catalyzing the dephosphorylation of several model phosphotase substrates, including p-nitrophenyl phosphate, tyrosine phosphorylated myelin basic protein and serine phosphorylated casein. A portion of this activity closely resembled dephosphorylation patterns of known protein tyrosine phosphatases. The reaction showed sensitivity to sodium orthovanadate, proceeded efficiently in the presence of metal chelators and favored acidic pH for optimum activity. Cell lysates from EGF-or isoproterenol-stimulated parotid glands, when immunoprecipitated with anti-Syp antibody, showed the induction of protein tyrosine phosphatase activity significantly higher than the unstimulated controls. The protein of M r = 65kDa also had elevated levels of tyrosine phosphorylation following isolation from cells treated to undergo proliferation. Thus parotid gland acinar cells possess protein tyrosine phosphatase activity of the PTPase 1D class associated with inducible cell growth, in addition to other phosphatases.

The purification and characterization of an extremely thermostable alpha-amylase from the hyperthermophilic archaebacterium Pyrococcus furiosus.

The alpha-amylase from Pyrococcus furiosus, a hyperthermophilic archaebacterium, has been purified to homogeneity. The enzyme is a homodimer with a subunit molecular mass of 66 kDa. The isoelectric point is 4.3. The enzyme displays optimal activity, with substantial thermal stability, at 100 degrees C, with the onset of activity at approximately 40 degrees C. Unlike mesophilic alpha-amylases there is no dependence on Ca2+ for activity or thermostability. The enzyme displays a broad range of substrate specificity, with the capacity to hydrolyze carbohydrates as simple as maltotriose. No subtrate binding occurs below the temperature threshold of activity, and a decrease in Km accompanies an increase in temperature. Except for a decrease in Asp and an increase in Glu, the amino acid composition does not confirm previously defined trends in thermal adaption. Fourth derivative UV spectroscopy and intrinsic fluorescence measurements detected no temperature-dependent structural reorganization. Hydrogen exchange results indicate that the molecule is rigid, with only a slight increase in conformational flexibility at elevated temperature. Scanning microcalorimetry detected no considerable change in the heat capacity function, at the pH of optimal activity, within the temperature range in which activity is induced. The heat absorption peak due to denaturation, under these conditions, occurred within the temperature range of 90-120 degrees C. When the pH was increased, a change in the shape of the heat absorption peak was observed, which when analyzed thermodynamically shows that the process of heat denaturation is complex, and includes at least three stages, indicating that the protein structure consists of three domains. At temperatures below 90 degrees C no excess heat absorption or change in the CD spectra were observed which could be associated with the cooperative conformational transition of the protein. According to the thermodynamic characteristics of the heat denaturation, the cold denaturation of this protein can be expected only at -3 degrees C. Therefore, the observed inactivation of this enzyme is not caused by the cooperative change of its tertiary structure. It can be associated only with the gradual changes of protein domain interaction.