Adaptation of a thermophilic enzyme, 3-isopropylmalate dehydrogenase, to low temperatures

Random mutagenesis coupled with screening of the active enzyme at a low temperature was applied to isolate cold-adapted mutants of a thermophilic enzyme. Four mutant enzymes with enhanced specific activities (up to 4.1-fold at 40 degrees C) at a moderate temperature were isolated from randomly mutated Thermus thermophilus 3-isopropylmalate dehydrogenase. Kinetic analysis revealed two types of cold-adapted mutants, i.e. k(cat)-improved and K(m)-improved types. The k(cat)-improved mutants showed less temperature-dependent catalytic properties, resulting in improvement of k(cat) (up to 7.5-fold at 40 degrees C) at lower temperatures with increased K(m) values mainly for NAD. The K(m)-improved enzyme showed higher affinities toward the substrate and the coenzyme without significant change in k(cat) at the temperatures investigated (30-70 degrees C). In k(cat)-improved mutants, replacement of a residue was found near the binding pocket for the adenine portion of NAD. Two of the mutants retained thermal stability indistinguishable from the wild-type enzyme. Extreme thermal stability of the thermophilic enzyme is not necessarily decreased to improve the catalytic function at lower temperatures. The present strategy provides a powerful tool for obtaining active mutant enzymes at lower temperatures. The results also indicate that it is possible to obtain cold-adapted mutant enzymes with high thermal stability.     

Functional significance of isoenzymes in thermal acclimatization. Acetylcholinesterase from trout brain

1. The effects of acclimatization temperature on the catalytic properties of acetylcholinesterase from rainbow-trout brain were examined. 2. Trout brain acetylcholinesterase occurs in two distinct forms. A single ;warm' variant of the enzyme is present after acclimatization to 17 degrees C; a single ;cold' variant appears after acclimatization to 2 degrees C. Both forms are present in fish after acclimatization to an intermediate temperature. 3. The K(m) values of the enzyme variants for acetylcholine are temperature-dependent, the lowest values coinciding with the acclimatization temperature at which each enzyme was induced. 4. It is concluded that the K(m)-temperature relationship is adaptive, and that the critical process during thermal acclimatization, in cases where enzymes show sharp changes in K(m) with temperature, is the synthesis of a new enzyme variant that is better suited for catalysis and control of catalysis under the conditions of the acclimatized state.     

Heat effect on the structure and activity of the recombinant glutamate dehydrogenase from a hyperthermophilic archaeon Pyrococcus horikoshii

Glutamate dehydrogenase from Pyrococcus horikoshii (Pho-GDH) was cloned and overexpressed in Escherichia coli. The cloned enzyme with His-tag was purified to homogeneity by affinity chromatography and shown to be a hexamer enzyme of 290+/-8 kDa (subunit mass 48 kDa). Its optimal pH and temperature were 7.6 and 90 degrees C, respectively. The purified enzyme has outstanding thermostability (the half-life for thermal inactivation at 100 degrees C was 4 h). The enzyme shows strict specificity for 2-oxoglutarate and L-glutamate and requires NAD(P)H and NADP as cofactors but it does not reveal activity on NAD as cofactor. K(m) values of the recombinant enzyme are comparable for both substrates: 0.2 mM for L-glutamate and 0.53 mM for 2-oxoglutarate. The enzyme was activated by heating at 80 degrees C for 1 h, which was accompanied by the formation of its active conformation. Circular dichroism and fluorescence spectra show that the active conformation is heat-inducible and time-dependent.     

Thr373, Asp375, and Lys260 are in the coenzyme site of porcine NADP-dependent isocitrate dehydrogenase

Thr(373), Lys(374), Asp(375), and Lys(260) were chosen as site-directed mutagenesis targets within porcine NADP-dependent isocitrate dehydrogenase based on structurally corrected sequence alignment among prokaryotic and eukaryotic NADP-isocitrate dehydrogenases. Wild-type and all mutant enzymes were expressed in Escherichia coli and purified to homogeneity. These mutations do not alter the secondary structure or dimerization state of the mutants. The D375N and K260Q mutants exhibit, respectively, a 15- and 28-fold increase in K(m) for NADP, along with marked decreases in V(max) as compared to wild-type enzyme. In contrast, replacing Lys(374), which was previously proposed to contribute to apparent coenzyme affinity, does not change the enzyme's kinetic parameters. T373S exhibits similar kinetic parameters to those of wild-type while T373A and T373V mutations reduce the V(max) values of the resulting enzymes to 1 and 20%, respectively of that of wild-type. We conclude that a hydroxyl group at position 373 is required for effective enzyme function and that Asp(375) and Lys(260) are critical amino acids contributing to coenzyme affinity as well as catalysis by porcine NADP-isocitrate dehydrogenase.     

Effect of environmental temperature on the kinetic properties of goldfish brain choline acetyltransferase

1. Michaelis constants of goldfish brain choline acetyltransferase were found to depend on the concentration of the second substrate present and on the temperature to which the fish had been adapted. 2. Primary plots constructed from results obtained with enzyme prepared from cold-adapted or warm-adapted fish indicated that synthesis of acetylcholine took place by a sequential mechanism. 3. The affinity of choline acetyltransferase for acetyl-CoA was about 100 times that for choline irrespective of whether the enzyme had been prepared from warm-adapted or cold-adapted fish. 4. The maximum rate at which choline acetyltransferase synthesized acetylcholine and the energy of activation for this synthesis remained independent of the previous environmental temperature of the fish. 5. The affinity of choline acetyltransferase for choline and acetyl-CoA showed a complex dependence on temperature. The affinity of the enzyme from cold-adapted fish for substrates increased as the incubation temperature was lowered, whereas that of the enzyme from warm-adapted fish first increased and then decreased. 6. The maximum affinity of choline acetyltransferase for both substrates, from both cold-adapted and warm-adapted fish, occurred at temperatures that corresponded approximately to the respective environmental temperatures of the fish. 7. These changes in enzyme affinity for substrates are not thought to be due to the presence of isoenzymes. Their adaptive significance is unknown, but it could be connected with the maintenance of the enzyme in a stable form.     

Evaluation by site-directed mutagenesis of aspartic acid residues in the metal site of pig heart NADP-dependent isocitrate dehydrogenase

Pig heart NADP-dependent isocitrate dehydrogenase requires a divalent metal cation for catalysis. On the basis of affinity cleavage studies [Soundar and Colman (1993) J. Biol. Chem. 268, 5267] and analysis of the crystal structure of E. coli NADP-isocitrate dehydrogenase [Hurley et al. (1991) Biochemistry 30, 8671], the residues Asp(253), Asp(273), Asp(275), and Asp(279) were selected as potential ligands of the divalent metal cation in the pig heart enzyme. Using a megaprimer PCR method, the Asp at each of these positions was mutated to Asn. The wild-type and mutant enzymes were expressed in Escherichia coli and purified. D253N has a specific activity, K(m) values for Mn(2+), isocitrate, and NADP, and also a pH-V(max) profile similar to those of the wild-type enzyme. Thus, Asp(253) is not involved in enzyme function. D273N has an increased K(m) for Mn(2+) and isocitrate with a specific activity 5% that of wild type. The D273N mutation also prevents the oxidative metal cleavage seen with Fe(2+) alone in the wild-type enzyme. As compared to wild type, D275N has greatly increased K(m) values for Mn(2+) and isocitrate, with a specific activity <0.1% that of wild type, and a large increase in pK(a) for the enzyme-substrate complex. D279N has only small increases in K(m) for Mn(2+) and isocitrate, but a specific activity <0.1% that of wild type and a major change in the shape of its pH-V(max) profile. These results suggest that Asp(273) and Asp(275) contribute to metal binding, whereas Asp(279), as well as Asp(275), is critical for catalysis. Asp(279) may function as the catalytic base. Using the Modeler program of Insight II, a structure for porcine NADP-isocitrate dehydrogenase was built based on the X-ray coordinates of the E. coli enzyme, allowing visualization of the metal-isocitrate site.     

Functional Determinants of Temperature Adaptation in Enzymes of Cold- versus Warm-Adapted Mussels (Genus Mytilus)

Temperature is a strong selective force on the evolution of proteins due to its effects on higher orders of protein structure and, thereby, on critical protein functions like ligand binding and catalysis. Comparisons among orthologous proteins from differently thermally adapted species show consistent patterns of adaptive variation in function, but few studies have examined functional adaptation among multiple structural families of proteins. Thus, with our present state of knowledge, it is difficult to predict what fraction of the proteome will exhibit adaptive variation in the face of temperature increases of a few to several degrees Celsius, that is, temperature increases of the magnitude predicted by models of global warming. Here, we compared orthologous enzymes of the warm-adapted Mediterranean mussel Mytilus galloprovincialis and the cold-adapted Mytilus trossulus, a native of the North Pacific Ocean, species whose physiologies exhibit significantly different responses to temperature. We measured the effects of temperature on the kinetics (Michaelis-Menten constant-K(m)) of five enzymes that are important for ATP generation and that represent distinct protein structural families. Among phosphoglucomutase (PGM), phosphoglucose isomerase (PGI), pyruvate kinase (PK), phosphoenolpyruvate carboxykinase (GTP) (PEPCK), and isocitrate dehydrogenase (NADP) (IDH), only IDH orthologs showed significantly different thermal responses of K(m) between the two species. The K(m) of isocitrate of M. galloprovincialis-IDH was intrinsically lower and more thermally stable than that of M. trossulus-IDH and thus had higher substrate affinity at high temperatures. Two amino acid substitutions account for the functional differences between IDH orthologs, one of which allows for more hydrogen bonds to form near the mobile region of the active site in M. galloprovincialis-IDH. Taken together, our findings cast light on the targets of adaptive evolution in the context of climate change; only a minority of proteins might adapt to small changes in temperature, and these adaptations may involve only small changes in sequence.     

Nicotinamide Adenine Dinucleotide Phosphate-Dependent Formate Dehydrogenase from Clostridium thermoaceticum: Purification and Properties

The nicotinamide adenine dinucleotide phosphate (NADP)-dependent formate dehydrogenase in Clostridium thermoaceticum used, in addition to its natural electron acceptor, methyl and benzyl viologen. The enzyme was purified to a specific activity of 34 (micromoles per minute per milligram of protein) with NADP as electron acceptor. Disc gel electrophoresis of the purified enzyme yielded two major and two minor protein bands, and during centrifugation in sucrose gradients two components of apparent molecular weights of 270,000 and 320,000 were obtained, both having formate dehydrogenase activity. The enzyme preparation catalyzed the reduction of riboflavine 5'-phosphate flavine adenine dinucleotide and methyl viologen by using reduced NADP as a source of electrons. It also had reduced NADP oxidase activity. The enzyme was strongly inhibited by cyanide and ethylenediaminetetraacetic acid. It was also inhibited by hypophosphite, an inhibition that was reversed by formate. Sulfite inhibited the activity with NADP but not with methyl viologen as acceptor. The apparent K(m) at 55 C and pH 7.5 for formate was 2.27 x 10(-4) M with NADP and 0.83 x 10(-4) with methyl viologen as acceptor. The apparent K(m) for NADP was 1.09 x 10(-4) M and for methyl viologen was 2.35 x 10(-3) M. NADP showed substrate inhibition at 5 x 10(-3) M and higher concentrations. With NADP as electron acceptor, the enzyme had a broad pH optimum between 7 and 9.5. The apparent temperature optimum was 85 C. In the absence of substrates, the enzyme was stable at 70 C but was rapidly inactivated at temperatures above 73 C. The enzyme was very sensitive to oxygen but was stabilized by thiol-iron complexes and formate.     

beta-Sulfur substituted alpha-ketoglutarates as inhibitors and alternate substrates for isocitrate dehydrogenases and certain other enzymes

The RS-isomers of beta-mercapto-alpha-ketoglutarate, beta-methylmercapto-alpha-ketoglutarate and beta-methylmercapto-alpha-hydroxyglutarate have been synthesized. Beta-Mercapto-alpha-ketoglutarate was a potent inhibitor, competitive with isocitrate and noncompetitive with NADP+, of the mitochondrial NADP-specific isozyme from pig heart (Ki = 5 nM; Km (DL-isocitrate)/Ki(RS-beta-mercapto-alpha-ketoglutarate) = 650) and pig liver, the cytosolic isozyme from pig liver (I0.5 = 23 nM), and the NADP-linked enzymes from yeast (Ki = 58 nM) and Escherichia coli (Ki = 58 nM) at pH 7.4 and with Mg2+ as activator. beta-Mercapto-alpha-ketoglutarate was also an effective inhibitor of NADP-isocitrate-dehydrogenase activity in intact liver mitochondria. beta-Mercapto-alpha-ketoglutarate was a much less potent inhibitor for heart NAD-isocitrate dehydrogenase (Ki = 520 nM) than for the NADP-specific enzyme. beta-Methylmercapto-alpha-ketoglutarate (I0.5 = 10 microM) was a much less effective inhibitor than the beta-mercapto derivative for heart NADP-isocitrate dehydrogenase. The beta-sulfur substituted alpha-ketoglutarates were substrates for the oxidation of NADPH by heart NADP-isocitrate dehydrogenase without requiring CO2. beta-Methylmercapto-alpha-hydroxyglutarate, the expected product of reduction of beta-methylmercapto-alpha-ketoglutarate, did not cause reduction of NADP+ but it was an inhibitor competitive with isocitrate for NADP-isocitrate dehydrogenase. The beta-sulfur substituted alpha-ketoglutarate derivatives were alternate substrates for alpha-ketoglutarate dehydrogenase and the cytosolic and mitochondrial isozymes of heart aspartate aminotransferase but had no effect on glutamate dehydrogenase or alanine aminotransferase.     

The acute regulation of mitochondrial proton conductance in cells and mitochondria from the brown fat of cold-adapted and warm-adapted guinea pigs

Cells and mitochondria were prepared from the brown adipose tissue of adult guinea-pigs adapted to either 4-7 degrees C or 22-25 degrees C. The cold-adapted cells displayed noradrenaline-stimulated, propranolol-sensitive respiration, but noradrenaline failed to increase the respiration of the warm-adapted cells. Purine-nucleotide-sensitive proton conductance was greater in cold-adapted mitochondria than in warm-adapted controls. At the same time cold-adapted mitochondria were extremely sensitive to the uncoupling effect of endogenous and infused fatty acids, and resembled the mitochondria from the brown adipose tissue of cold-adapted hamsters. Warm-adapted mitochondria were ninefold less sensitive, and resembled liver mitochondria. With cold-adapted, but not warm-adapted mitochondria, respiration increased proportionately to the rate of fatty acid infusion. It is concluded that the presence of the 32000-Mr proton conductance pathway is necessary for the expression of a high sensitivity to fatty acid uncoupling, suggesting that the fatty acids interact directly with this protein to modulate the proton conductance during the acute regulation of thermogenesis.     

Kinetic models for synthesis by a thermophilic alcohol dehydrogenase

Alcohol dehydrogenase (E. C. 1.1.1.1) from Thermoanaerobium brockii at 25 degrees C and at 65 degrees C is more active with secondary than primary alcohols. The enzyme utilizes NADP and NADPH as cosubstrates better than NAD and NADH. The maximum velocities (V(m)) for secondary alcohols at 65 degrees C are 10 to 100 times higher than those at 25 degrees C, whereas the K(m) values are more comparable.At both 25 degrees C and 65 degrees C the substrate analogue 1,1,1,3,3,3-hexafluoro-2-propanol inhibited the oxidation of alcohol competitively with respect to cyclopentanol, and uncompetitively with respect to NADP. Dimethylsulfoxide inhibited the reduction of cyclopentanone competitively with respect to cyclopentanone, and uncompetitively with respect to NADPH. As a product inhibitor, NADP was competitive with respect to NADPH. These results demonstrate that the enzyme binds the nucleotide and then the alcohol or ketone to form a ternary complex which is converted to a product ternary complex that releases product and nucleotide in that order.At 25 degrees C, all aldehydes and ketones examined inhibited the enzyme at concentrations above their Michaelis constants. The substrate inhibition by cyclopentanone was incomplete, and it was uncompetitive with respect to NADPH. Furthermore, cyclopentanone as a product inhibitor showed intercept-linear, slope-parabolic inhibition with respect to cyclopentanol. These results indicate that cyclopentanone binds to the enzyme-NADP complex at high concentrations. The resulting ternary complex slowly dissociates NADP and cyclopentanone.At 65 degrees C, all of the secondary alcohols, with the exception of cyclohexanol, show substrate activation at high concentration. Experiments in which NADP was the variable substrate and cyclopentanol as the constant-variable substrate over a wide range of concentrations gave double reciprocal plots in which the intercepts showed substrate activation and the slopes showed substrate inhibition. These results indicate that the secondary alcohols bind to the enzyme-NADPH complex at high concentrations and that the resulting ternary complex dissociates NADPH faster than the enzyme-NADPH complex. (c) 1993 John Wiley & Sons, Inc.     

Properties of the apo-form of the NADP(H)-binding domain III of proton-pumping Escherichia coli transhydrogenase: implications for the reaction mechanism of the intact enzyme

Proton-translocating nicotinamide nucleotide transhydrogenases contain an NAD(H)-binding domain (dI), an NADP(H)-binding domain (dIII) and a membrane domain (dII) with the proton channel. Separately expressed and isolated dIII contains tightly bound NADP(H), predominantly in the oxidized form, possibly representing a so-called "occluded" intermediary state of the reaction cycle of the intact enzyme. Despite a K(d) in the micromolar to nanomolar range, this NADP(H) exchanges significantly with the bulk medium. Dissociated NADP(+) is thus accessible to added enzymes, such as NADP-isocitrate dehydrogenase, and can be reduced to NADPH. In the present investigation, dissociated NADP(H) was digested with alkaline phosphatase, removing the 2'-phosphate and generating NAD(H). Surprisingly, in the presence of dI, the resulting NADP(H)-free dIII catalyzed a rapid reduction of 3-acetylpyridine-NAD(+) by NADH, indicating that 3-acetylpyridine-NAD(+) and/or NADH interacts unspecifically with the NADP(H)-binding site. The corresponding reaction in the intact enzyme is not associated with proton pumping. It is concluded that there is a 2'-phosphate-binding region in dIII that controls tight binding of NADP(H) to dIII, which is not a required for fast hydride transfer. It is likely that this region is the Lys424-Arg425-Ser426 sequence and loops D and E. Further, in the intact enzyme, it is proposed that the same region/loops may be involved in the regulation of NADP(H) binding by an electrochemical proton gradent.     

Properties of Nicotinamide Adenine Dinucleotide Phosphate-Dependent Formate Dehydrogenase from Clostridium thermoaceticum

Li, Lan-Fun (Western Reserve University School of Medicine, Cleveland, Ohio), Lars Ljungdahl, and Harland G. Wood. Properties of nicotinamide adenine dinucleotide phosphate-dependent formate dehydrogenase from Clostridium thermoaceticum. J. Bacteriol. 92: 405-412. 1966.-A nicotinamide adenine dinucleotide phosphate (NADP)-dependent formate dehydrogenase has been isolated from C. thermoaceticum. The enzyme is very sensitive to oxygen and requires sulfhydryl compounds for activity. The apparent K(m) at 50 C and pH 7.0 for NADP is 5.9 x 10(-5)m and for formate, 2.2 x 10(-4)m. The enzyme is most active at about 60 C and at pH values between 7.0 and 9.0. The enzyme catalyzes an exchange between C(14)O(2) and formate, which requires NADP, but net synthesis of formate from CO(2) and reduced nicotinamide adenine dinucleotide phosphate could not be demonstrated. The reaction does not involve ferredoxin.     

Kinetic properties of glucose-6-phosphate dehydrogenase from lamb kidney cortex

Glucose-6-phosphate dehydrogenase is the key regulatory enzyme of the pentose phosphate pathway and one of the products of this enzyme; NADPH has a critical role in the defence system against the free radicals. In this study, glucose-6-phosphate dehydrogenase from lamb kidney cortex kinetic properties is examined. The purification procedure is composed of two steps after ultracentrifugation for rapid and easy purification: 2', 5'-ADP Sepharose 4B affinity and DEAE Sepharose Fast Flow anion exchange chromatography. Previously, we used this procedure for the purification of glucose-6-phosphate dehydrogenase from bovine lens. The double reciprocal plots and product inhibition studies showed that the enzyme obeys 'Ordered Bi Bi' mechanism: K(m NADP+)K(m G-6-P) and K(i G-6-P) (dissociation constant of the enzyme--G-6-P complex) were found to be 0.018 +/- 0.002, 0.039 +/- 0.006 and 0.029 +/- 0.005 mM, respectively, by using nonlinear regression analysis. The enzyme was stable at 4 degrees C for a week.     

Occurrence of a novel NADP(+)-linked alcohol dehydrogenase in Euglena gracilis

An NADP(+)-dependent alcohol dehydrogenase was found in Euglena gracilis Z grown on 1-hexanol, while it was detected at low activity in cells grown on ethanol or glucose as a carbon source, indicating that the enzyme is induced by the addition of 1-hexanol into the medium as a carbon source. This enzyme was extremely unstable, even at 4 degrees C, unless 20% ethylene glycol was added. The optimal pH was 8.8-9.0 for oxidation reaction. The apparent K(m) values for 1-hexanol and NADP(+) were found to be 6.79 mM and 46.7 microM for this enzyme, respectively. The substrate specificity of this enzyme was very different from that of already purified NAD(+)-specific ethanol dehydrogenase by showing the highest activity with 1-hexanol as a substrate, followed by 1-pentanol and 1-butanol, and there was very little activity with ethanol and 1-propanol. This enzyme was active towards the primary alcohols but not secondary alcohols. Accordingly, since the NADP(+)-specific enzyme was separated on DEAE cellulose column, Euglena was confirmed to contain a novel enzyme to be active towards middle and long-chain length of fatty alcohols.     

Purification and characterization of a cold-adapted isocitrate lyase and expression analysis of the cold-inducible isocitrate lyase gene from the psychrophilic bacterium<Emphasis Type="Italic"> Colwellia psychrerythraea</Emphasis>

Isocitrate lyase (ICL) from Colwellia psychrerythraea, a psychrophilic bacterium, was purified and characterized. The subunit molecular mass was 64 kDa, which is larger than that of other bacterial ICLs. The optimal temperature for its activity was 25 degrees C, the value of K(m) for the substrate ( DL-isocitrate) was minimum at 15 degrees C, and the catalytic efficiency ( k(cat)/ K(m)) value was maximum at 20 degrees C. Furthermore, the enzyme was remarkably thermolabile and completely inactivated by incubation for 2 min at 30 degrees C. These features indicate that ICL from this bacterium is a typical cold-adapted enzyme. A partial amino acid sequence of the C. psychrerythraea ICL was very similar to that of the closely related psychrophile Colwellia maris. Expression of the gene encoding the C. psychrerythraea ICL was found to be induced by low temperatures and by acetate in the medium. The cold adaptation of the catalytic properties of ICL and the stimulated expression of its gene at low temperatures strongly suggest that this enzyme is important for the growth of this bacterium in a cold environment.     

Temperature dependence of kinetic parameters for hyperthermophilic glutamate dehydrogenase from Aeropyrum pernix K1

The temperature dependence of the steady-state kinetic parameters for a glutamate dehydrogenase from Aeropyrum pernix K1 was investigated. The enzyme showed a biphasic kinetic characteristic for L-glutamate and a monophasic one for NADP at 50-90 degrees C. At low concentrations of L-glutamate the Km decreased from 2.02 to 0.56 mM and the catalytic efficiency (Vmax/Km) markedly increased (4-150 micromol x mg(-1) x mM(-1)) along with the increase of temperature from 50 to 90 degrees C. At high concentrations of the substrate the Km was fairly high and approximately constant (around 225 mM), and the catalytic efficiency was low and its temperature-dependent change was small. The Km (0.039 mM) for NADP did not change with the increase of temperature. In the reductive amination, the Kms for 2-oxoglutarate (1.81 and 9.37 mM at low and high levels of ammonia, respectively) were independent on temperature, but the Kms for ammonia and NADPH rose from 86 to 185 mM and 0.050 to 0.175 mM, respectively.     

A multilocus system for studying tissue and subcellular specialization. The pH and temperature dependence of the two major NADP-dependent isocitrate dehydrogenase isozymes of the fish Fundulus heteroclitus

In the teleost fish Fundulus heteroclitus, there are three NADP-dependent isocitrate dehydrogenase isozymes. IDH-B2 is the only cytoplasmic isozyme, and IDH-C2 dominates the mitochondria of all tissues other than liver, where IDH-A2 is expressed. Since fish are ectotherms, their intracellular temperature and pH change directly with environmental temperature. In order to evaluate the influence of these environmental parameters on a model fish NADP-isocitrate dehydrogenase system, the major cytoplasmic (IDH-B2) and mitochondrial (IDH-C2) isozymes were kinetically evaluated as a function of pH and temperature. Whereas Vfmax and KmISOCm (where ISOC is isocitrate) were pH-independent, the Km for NADP was pH-dependent for both isozymes. The cytoplasmic isozyme (IDH-B2) had smaller KmNADP values between pH 7.0 and pH 8.0 than the mitochondrial form (IDH-C2). Vfmax and Km for substrate and coenzyme were temperature-dependent. Energy of activation for IDH-B2 and IDH-C2 was 10.6 and 12.8 kcal/mol, respectively. Both proteins had delta G not equal to values of about 15.8 kcal/mol, with significantly different distributions between delta H not equal to and delta S not equal to. The cytoplasmic isozyme (IDH-B2) appears to have a greater rate of catalysis than the mitochondrial enzyme (IDH-C2) at temperatures less than 30 degrees C. Moreover, the IDH-B2 isozyme had lower KmNADP values than the IDH-C2 isozyme at all temperatures, whereas the KmISOC values for the two isozymes were indistinguishable. Our data suggest that the two major NADP-dependent isocitrate dehydrogenase isozymes have unique physiological and metabolic functions that are adapted to the tissues and cellular compartments in which they are expressed.     

Use of gel electrophoresis for the study of enzymatic activities of cold-adapted bacteria

Glutamate dehydrogenase (GDH) and lactate dehydrogenase (LDH) activity of 13 cold-adapted strains, isolated from cold soils and showing GDH and/or LDH activity in spectrophotometric assays, were revealed by the use of electrophoresis on a nondenaturing acrylamide gel (zymogram). Psychrophilic strains were grown at 4 degrees C and 10 degrees C and the psychrotolerant strains at 4 degrees, 20 degrees and 28 degrees C. Incubation with the specific substrate and staining were done at 4, 28 or 37 degrees C. In the most cold-adapted strains, LDH and GDH production was high at 4 degrees C. In psychrotrophic strains, enzyme production and activity were greater at 20 or 28 degrees C than at lower temperatures. LDH remained active up to 37 degrees C while GDH activity was more thermolabile. GDH activity was NAD-dependent in some psychrophilic strains. In other strains, it was dependent on NAD(P) only or on both NAD and NAD(P). Two bands were seen for GDH or LDH activity in some strains. This method, which does not require a dialysis step, can be used to study the influence of temperature on enzyme production and activity, and the co-factor dependence. It detects phenotypic differences between isozymes, providing data for systematics.     

The artificial evolution of an enzyme by random mutagenesis: the development of formaldehyde dehydrogenase

A unique variant of glutathione independent formaldehyde dehydrogenase of Pseudomonas putida was obtained by random mutagenesis using the PCR-reaction. This YM042 mutant, S318G, was a cold-adapted formaldehyde dehyrogenase. The activity at 29 degrees C of the variant was 1.7-fold higher than that of the wild type. The K(m) values of the mutant at 37 degrees C were 0.40 mM for NAD(+) and 2.5 mM for formaldehyde, while those of the wild-type were 0.18 mM for NAD(+) and 2.1 mM for formaldehyde. The catalytic efficiency for formaldehyde was about 1.5-fold greater in the mutant than in the wild-type enzyme. The optimum pHs and temperatures of the mutant and the wild-type enzyme were 7.5, and 8.0 and 37 degrees C, and 47 degrees C, respectively. The thermal stability of the mutant was lower than that of the wild type.