Kinetic Analysis in Mixed Micelles of Partially Purified Rat Brain Phospholipase D Activity and its Activation by Phosphatidylinositol 4,5-Bisphosphate

A partially purified rat brain membrane phospholipase D (PLD) activity was characterized in a mixed micellar system consisting of l-palmitoyl-2-[6-N-(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)-amino]caproyl-phosphatidylcholine (NBD-PC) and Triton X-100, under conditions where Triton X-100 has a surface dilution effect on PLD activity and the catalytic rate is dependent on the surface concentration (expressed in terms of molar ratio) of NBD-PC. PLD activity was specifically activated by phosphatidylinositol 4,5-bisphosphate (PIP₂), and the curve of activation versus PIP₂ molar ratio fitted a Michaelis-Menten equation with a K_act value between molar ratios of 0.001–0.002. Maximal activation was observed at a PIP₂ molar ratio of 0.01. Similar values were obtained when activities of partially purified PLD as well as membrane-bound PLD were determined towards pure NBD-PC micelles. In the mixed micellar system PIP₂ was shown to elevate by 6–22 fold the specificity constant of PLD towards NBD-PC (K_A, which is proportional to V_max/K_m). Kinetic analysis of PLD trans-phosphatidylation activity towards ethanol, 1-propanol and 1-butanol revealed a Michaelis-Menten type dependence on alcohol concentration up to 1000, 200 and 80 mM, respectively. While V_max values were similar towards all three alcohols, enzyme affinity increased as the alcohol was longer, and K_m values for ethanol, 1-propanol and 1-butanol were 291, 75 and 16 mM (respectively). PLD specificity constants (K_A) towards ethanol, 1-propanol and 1-butanol were shown to be respectively 260, 940 and 5,920 times higher than to water, the competing substrate. 1-Propanol and 1-butanol inhibited PLD activity above 400 and 100 mM, respectively. The present results indicate that partially purified PLD obeys surface dilution kinetics with regard to its phospholipid substrate PC and its cofactor PIP₂, and that in the presence of alcohols, its transphosphatidylation activity may be analyzed as a competitive reaction to the hydrolysis reaction.     

Activation and inhibition of mitochondrial adenosine triphosphatase by various anions and other agents

The activating or inhibiting actions of a variety of anion species and of oligomycin, aurovertin and Dio-9 on the ATPase of a sonic particle preparation of rat liver mitochondria have been characterized by measurements of the relevantV _max,K _i andK _m values.The normalV _max was increased by a factor near 7 by the anions: dichromate, chromate, pyrophosphate, orthophosphate, orthoarsenate and sulphate. The fully activating concentration varied from about 2 mM for dichromate to 150 mM for sulphate. The increase inV _max was accompanied by a time-dependent decrease in (K _i)ADP, but there was no change in (K _m)ATP. The increase inV _max by the activating anions was abolished by aurovertin; but in presence of oligomycin, the lowV _max was increased by the activating anions by the same factor as theV _max in absence of oligomycin.Certain anions, notably azide, decreasedV _max, but did not affect (K _i)ADP or (K _m)ATP. The decrease inV _max by azide and oligomycin were approximately additive. Even at high concentration, Dio-9 was without detectable effect on the ATPase, but it had a gramicidinlike effect on the intact mitochondria.The specificity of the ATPase for ATP relative to GTP was found to be attributable to the high value of (V _max)ATP compared with (V _max)GTP. The values of (K _m)ATP and (K _m)GTP were virtually the same.Some rationalization of these and other supporting observations is attempted in terms of present knowledge of the constitution of the ATPase complex.     

Temperature sensitivity of soil enzyme kinetics under N‐fertilization in two temperate forests

Soil microbes produce extracellular enzymes that degrade carbon (C)‐containing polymers in soil organic matter. Because extracellular enzyme activities may be sensitive to both increased nitrogen (N) and temperature change, we measured the effect of long‐term N addition and short‐term temperature variation on enzyme kinetics in soils from hardwood forests at Bear Brook, Maine, and Fernow Forest, West Virginia. We determined the V_max and K_m parameters for five hydrolytic enzymes: α‐glucosidase, β‐glucosidase, β‐xylosidase, cellobiohydrolase, and N‐acetyl‐glucosaminidase. Temperature sensitivities of V_max and K_m were assessed within soil samples subjected to a range of temperatures. We hypothesized that (1) N additions would cause microbial C limitation, leading to higher enzyme V_max values and lower K_m values; and (2) both V_max and K_m would increase at higher temperatures. Finally, we tested whether or not temperature sensitivity of enzyme kinetics is mediated by N addition. Nitrogen addition significantly or marginally significantly increased V_max values for all enzymes, particularly at Fernow. Nitrogen fertilization led to significantly lower K_m values for all enzymes at Bear Brook, but variable K_m responses at Fernow Forest. Both V_max and K_m were temperature sensitive, with Q₁₀ values ranging from 1.64–2.27 for enzyme V_max and 1.04–1.93 for enzyme K_m. No enzyme showed a significant interaction between N and temperature sensitivity for V_max, and only β‐xylosidase showed a significant interaction between N and temperature sensitivity for K_m. Our study is the first to experimentally demonstrate a positive relationship between K_m and temperature for soil enzymes. Higher temperature sensitivities for V_max relative to K_m imply that substrate degradation will increase with temperature. In addition, the V_max and K_m responses to N indicate greater substrate degradation under N addition. Our results suggest that increasing temperatures and N availability in forests of the northeastern US will lead to increased hydrolytic enzyme activity, despite the positive temperature sensitivity of K_m.     

A new alcohol dehydrogenase with unique stereospecificity from Pseudomonas sp.

A new nicotinamide cofactor-dependent alcohol dehydrogenase from Pseudomonas strain SBD6 (PADH) was isolated and purified 150-fold to homogeneity using a combination of salt precipitation, anion-exchange chromatography, gel filtration chromatography, and dye matrix chromatography. Approximately 10 mg of pure enzyme can be obtained from 10 g of wet cells. The enzyme has four subunits with a total molecular weight of 162,000. Incubation with the metal chelators 1,10-phenanthroline, 2-aminoethanethiol, hydroxyquinolinesulfonic acid, N-ethylmaleimide, and potassium cyanide result in complete loss of activity. The enzyme is very stable (t_1/2 7 days at pH 7 and 25°C in the absence of 2-propanol and 18 days in the presence of 10% 2-propanol, v/v) and possesses a broad substrate specificity with transfer of the pro-(R) hydride from NADH to the si face of carbonyl substrates to give (R)-alcohols in high enantiomeric excess, a stereochemical process different from that of other known alcohol dehydrogenases. Synthetic scale reductions are facilitated with 2-propanol as a hydride source for the regeneration of NADH. The kinetic mechanism is ordered bi-bi with the cofactor binding first. Based on NAD and 2-propanol, the kinetic parameters of the enzyme were determined to be V_max = 29.9 Units mg⁻¹ at 25°C and pH 8.5, K_m^NAD = 0.36 m and K_m^2-propanol = 0.19 m .     

The uptake of serotonin and dopamine by homogenates of frozen rat and human brain tissue

A recently described procedure of freezing and thawing, which allows retention of metabolic and functional integrity, has been applied in the study of serotonin and dopamine uptake into frozen rat and post mortem human frozen tissue. TheK _m andV _max for the serotonin uptake into human hypothalamus were estimated to be 0.12 M and 0.03 nmol/g/min respectively. TheK _m andV _max for the dopamine uptake into human putamen were estimated to be 0.28 M and 0.13 nmol/g/min respectively. The results indicate that the freezing procedure does not affect the uptake sites for these transmitters. The storage time before freezing is however of importance for theV _max value. TheK _m value for the uptake, on the other hand, seems to be rather resistant to storage time before freezing.     

Temperature sensitivities of extracellular enzyme Vmax and Km across thermal environments

The magnitude and direction of carbon cycle feedbacks under climate warming remain uncertain due to insufficient knowledge about the temperature sensitivities of soil microbial processes. Enzymatic rates could increase at higher temperatures, but this response could change over time if soil microbes adapt to warming. We used the Arrhenius relationship, biochemical transition state theory, and thermal physiology theory to predict the responses of extracellular enzyme V_max and K_m to temperature. Based on these concepts, we hypothesized that V_max and K_m would correlate positively with each other and show positive temperature sensitivities. For enzymes from warmer environments, we expected to find lower V_max, K_m, and K_m temperature sensitivity but higher V_max temperature sensitivity. We tested these hypotheses with isolates of the filamentous fungus Neurospora discreta collected from around the globe and with decomposing leaf litter from a warming experiment in Alaskan boreal forest. For Neurospora extracellular enzymes, V_max Q₁₀ ranged from 1.48 to 2.25, and K_m Q₁₀ ranged from 0.71 to 2.80. In agreement with theory, V_max and K_m were positively correlated for some enzymes, and V_max declined under experimental warming in Alaskan litter. However, the temperature sensitivities of V_max and K_m did not vary as expected with warming. We also found no relationship between temperature sensitivity of V_max or K_m and mean annual temperature of the isolation site for Neurospora strains. Declining V_max in the Alaskan warming treatment implies a short‐term negative feedback to climate change, but the Neurospora results suggest that climate‐driven changes in plant inputs and soil properties are important controls on enzyme kinetics in the long term. Our empirical data on enzyme V_max, K_m, and temperature sensitivities should be useful for parameterizing existing biogeochemical models, but they reveal a need to develop new theory on thermal adaptation mechanisms.     

Potassium modulation of methionine uptake in astrocytes in vitro

Methionine participates in a large variety of metabolic pathways in brain, and its transport may play an important regulatory role. The properties of methionine uptake were examined in a preparation of neonatal rat brain astrocytes. Uptake is linear for 15 minutes, up to 2.5 M. At steady state conditions, methionine is concentrated 30–50-fold. Measured methionine homoexchange accounts for a significant fraction of uptake at concentrations greater than 10 M. We recently reported that methionine uptake is decreased by elevations in extracellular K⁺. Potassium induced efflux cannot account for this apparent effect; and thus for concentrations less than 2.5M, and for short times of incubation, measured rates of methionine uptake represent unidirectional flux. At extracellular concentrations of K⁺ equal to 6.9 mM, the apparentV _max of methionine transport is 182 pmol/min/mg protein, and theK _m is 1.3 M. Where K⁺ is shifted to 11.9 mM, theK _m remains unchanged, and theV _max is reduced by half.     

Thyroid Hormone-Induced Alterations in Membrane Structure-Function Relationships: Studies on Kinetic Properties of Rat Kidney Microsomal Na+,K+-ATPase and Lipid/Phospholipid Profiles

The effects of thyroidectomy (Tx) and subsequent treatment with 3,5,3′-triiodothyronine (T₃) or combined replacement therapy (T_R) with T₃ and thyroxine (T₄) on the substrate and temperature kinetics properties of Na⁺,K⁺-ATPase and lipid/phospholipid makeup of rat kidney microsomes were examined. Enzyme activity was somewhat high in the hypothyroid (Tx) animals and increased significantly following T₃ treatment, while T_R treatment caused a decrease. In the Tx and T₃ groups enzyme activity resolved in two kinetic components, while in the T_R group the enzyme showed allosteric behavior up to 0.5 mm ATP concentration. The K _m and V _max values of both the components decreased in Tx animals without affecting the catalytic efficiency. T₃ treatment caused a significant increase in the V _max of both the components, with a significant increase in the catalytic efficiency, while the K _m values were not upregulated. The T_R regimen lowered the K _m and V _max of component II but improved the catalytic efficiency. Thyroid status-dependent changes were also noted in the temperature kinetics of the enzyme. Regression analysis revealed that changes in the substrate and temperature kinetics parameters correlated with specific phospholipid components.     

Transport of thymidine during the cell cycle in mitotically synchronized CHO cells

The kinetics of total uptake of thymidine into the cell were determined for cells which had been mitotically synchronized, plated into scintillation vials and pulsed with five concentrations of [³H]-thymidine at various times during the cell cycle. From Lineweaver-Burk plots of these rates, V_max and K_m values were determined for the transport of thymidine. The V_max values ranged from a low of 2.0 pmoles/ min/10⁶ cells in mid-G1 to a high of 99.7 in mid-S before a decline in late S and G2. K_m values displayed only a 5-fold range in values.     

Multiple regulatory sites in the C-terminal autoinhibitory domain of the plasma membrane H+-ATPase

The H⁺-ATPase activities of root and leaf plasma membranes from tobacco (Nicotiana tabacum) have been characterized with respect to V_max, K_m for ATP, pH dependence and activation involving the C-terminal autoinhibitory domain. With root plasma membranes, addition of lysophosphatidylcholine (lyso-PC) resulted in the expected increase in V_max, a decrease in K_m(ATP), and a shift in pH optimum to a more alkaline pH, typical for activation via the C-terminal inhibitory domain. With leaf plasma membranes, however, K_m(ATP) was relatively low and the pH optimum was around pH 7.0 before the addition of lyso-PC and did not change upon addition of the activator, although V_max increased twofold. Similar results were obtained with the in vivo activator fusicoccin. The results obtained with the leaf plasma membranes show that V_max may be regulated independently of K_m(ATP) and pH optimum, and suggest the presence of at least two regulatory sites within the C-terminal autoinhibitory domain of the H⁺-ATPase.     

Effects of temperature and CO2 enrichment on kinetic properties of NADP+-malate dehydrogenase in two ecotypes of Barnyard grass (Echinochloa crus-galli (L.) Beauv.) from contrasting climates

Summary The apparent energy of activation (E _a), Michaelis-Menten constant (K _mfor oxaloacetate), V _max/K _mratios and specific activities of NADP⁺-malate dehydrogenase (NADP⁺-MDH; EC 1.1.1.82) were analyzed in plants of Barnyard grass from Québec (QUE) and Mississippi (MISS) acclimated to two thermoperiods 28/22°C, 21/15°C, and grown under two CO₂ concentrations, 350 l l^-1 and 675 l l^-1. E _avalues of NADP⁺-MDH extracted from QUE plants were significantly lower than those of MISS plants. K _mvalues and V _max/K _mratios of the enzyme from both ecotypes were similar over the range of 10–30°C but reduced V _max/K _mratios were found for the enzyme of QUE plants at 30 and 40°C assays. MISS plants had higher enzyme activities when measured on a chlorophyll basis but this trend was reversed when activities were expressed per fresh weight leaf or per leaf surface area. Activities were significantly higher in plants of both populations acclimated to 22/28°C. CO₂ enrichment did not modify appreciably the catalytic properties of NADP⁺-MDH and did not have a compensatory effect upon catalysis or enzyme activity under cool acclimatory conditions. NADP⁺-MDH activities were always in excess of the amount required to support observed rates of CO₂ assimilation and these two parameters were significantly correlated. The enhanced photosynthetic performance of QUE plants under cold temperature conditions, as compared to that of MISS plants, cannot be attributed to kinetic differences of NADP⁺-malate dehydrogenase among these ecotypes.     

Transport of glucose,fructose and sucrose by Streptanthus tortuosus suspension cells

Streptanthus tortuosus Kell. suspension cells will grow in a medium with sucrose as carbohydrate source. It was investigated whether the cells are able to take up sucrose or whether sucrose has to be hydrolyzed to glucose and fructose which eventually are taken up. The detailed quantitative analysis of sugar-uptake rates in the low concentration range up to 1 mM showed the following features: (i) There is definitely no sucrose-uptake system working in the low concentration range; any uptake of radioactivity from labelled sucrose proceeds via hydrolysis of sucrose by cell-wallbound invertase. (ii) Hexoses are taken up by two systems, a glucose-specific system with a K _m of 45 M and a high V _max for glucose and a K _m of 6 mM and a low V _max for fructose, and a fructosespecific system with a K _m of 500 M and high a V _max for fructose and a K _m of 650 M and a low V _max for glucose. (iii) There is a more than tenfold preference for uptake of the fructose derived from sucrose versus uptake of free fructose, with the result that the kinetic disadvantage of the fructoseuptake system compared to the glucose-uptake system is diminished if sucrose is supplied as the carbon source. It is speculated that invertase might work as an enzyme aiding in fructose transport.Abbreviations FCCP carbonylcyanide-p-trifluoromethoxyphenylhydrazone - FW fresh weight     

Imidazolium chloride‐based ionic liquid‐assisted improvement of lipase activity in organic solvents

The activity of a lipase from a newly isolated Pseudomonas sp. was investigated in the presence of organic solvents and imidazolium chloride‐based ionic liquids (IL) such as BMIM[Cl] and HMIM[Cl]. The lipase activity in the presence of IL was higher compared to that in common organic solvents such as methanol and 2‐propanol. A possible explanation for the enzyme activation might be the structural changes induced in the protein in organic systems. Since IL quench the intensity of fluorescence emission, it was not possible to investigate the major factor that influences the enzyme behavior in these new organic salts. Furthermore, the enzyme exhibited excellent activity in buffer mixtures containing both organic solvent and IL. The stability of the lipase at 50°C was considerably increased in the presence of 20% BMIM[Cl] compared with the untreated lipase in aqueous medium. The light scattering method clearly showed that prevention of aggregation could be the reason for thermal stabilization at 50°C in reactions containing IL. Kinetic analysis of the enzyme in the presence of different concentrations of IL showed that the K_m value increased from 0.45 mM in aqueous buffer to 2.4 mM in 50% v/v BMIM[Cl]/buffer. The increase in K_m indicates that IL can significantly reduce the binding affinity of the substrate to the enzyme. Also, a linear correlation was observed between the BMIM[Cl] concentration and V_max of the enzyme. As the concentration of BMIM[Cl] increased from 10 to 50% v/v, the V_max value increased from 1.8 to 46 μM/min.     

The influence of salinity on the kinetics of NH inf4 sup+ uptake in Spartina alterniflora

Summary The effects of short- and long-term exposure to a range in concentration of sea salts on the kinetics of NH _inf4 ^sup+ uptake by Spartina alterniflora were examined in a laboratory culture experiment. Long-term exposure to increasing salinity up to 50 g/L resulted in a progressive increase in the apparent K_m but did not significantly affect V_max (mean V_max=4.23±1.97 mole·g^–1·h^–1). The apparent K_m increased in a nonlinear fashion from a mean of 2.66±1.10 mole/L at a salinity of 5 g/L to a mean of 17.56±4.10 mole/L at a salinity of 50 g/L. These results suggest that the long-term effect of exposure to total salt concentrations within the range 5–50 g/L was a competitive inhibition of NH _inf4 ^sup+ uptake in S. alterniflora. No significant NH _inf4 ^sup+ uptake was observed in S. alterniflora exposed to 65 g/L sea salts. Short-term exposure to rapid changes in salinity significantly affected both V_max and K_m. Reduction of solution salinity from 35 to 5 g/L did not change V_max but reduced K_m by 71%. However, exposing plants grown at 5 g/L salinity to 35 resulted in an decrease in V_max of approximately 50%. Exposure of plants grown at 35 g/L to a total sea salt concentration of 50 g/L for 48h completely inhibited uptake of NH _inf4 ^sup+ . For both experiments, increasing salinity led to an increase in the apparent K_m similar to that found in response to long-term exposure. Our data are consistent with a conceptual model of changes in the productivity of S. alterniflora in the salt marsh as a function of environmental modification of NH _inf4 ^sup+ uptake kinetics.     

A study of the substrate specificity of aminopeptidase N from Lactococcus lactis subsp. cremoris Wg2

A systematic study was made of the ability of aminopeptidase N from Lactococcus lactis subsp. cremoris Wg2 to hydrolyse different peptide substrates. The enzyme showed a marked preference for substrates containing arginine as the N-terminal residue but, to a lesser extent, was also capable of cleaving other residues such as lysine and leucine. There was a tendency for the activity to increase with the hydrophobicity index of the C-terminal residue of dipeptide substrates. It was also observed that the enzyme tended to have higher affinities but lower V _max values for tripeptides with hydrophobic C-terminal residues. The values determined for K _m and V _max increased with chain length for oligopeptides of the general formula Lys-Phe-(Gly) _n , the optimum, as determined from V _max/K _m, being when n = 4. Typical K _m values for the most effective substrates were in the range 0.2–0.6 mM.     

The effect of prior exposure on sodium uptake in tadpoles exposed to low pH water

Summary Bullfrog tadpoles (Rana catesbeiana) were pre-exposed (7d) to 5 different solutions which varied in ionic composition and pH. Unidirectional sodium influx was then measured over a range of sodium concentrations (50–2000 M) and at 2 pH's (4.0 and 5.8). Tadpoles pre-treated in either distilled water or pH 4.0 soft water exhibited higher sodium influx than animals pre-treated in pH 5.8 soft water. Pre-exposure to solutions high in calcium or sodium reduced sodium influx. Tadpoles pre-treated in pH 4.0 soft water exhibited an increasedV _max for sodium transport, whileK _m was unchanged relative to tadpoles pre-treated in pH 5.8 soft water. In contrast,K _m was increased andV _max was unchanged in tadpoles pre-exposed to high concentrations of calcium or sodium. Within each pre-exposure treatment, influx was inhibited in pH 4.0 test water relative to pH 5.8 test water. However, the magnitude of inhibition was lessened with increasing external concentrations of sodium.Abbreviations ASW artificial soft water - DBM dry body mass - DW distilled water     

Seasonal pattern of ammonium (methylamine) uptake by phytoplankton in an oligotrophic lake

Our primary objective was to determine if a relationship existed between seasonal change in phytoplankton and high affinity for (K _m) or uptake rates (V _maX) of ammonium which might explain seasonal phytoplankton succession in oligotrophic ecosystems. We measured ammonium uptake using [¹⁴C]-methylamine and estimatedK _m andV _max using Hanes Plots at 2-week intervals during 6 months of thermal stratification in Mountain lake, Virginia (37° 22 N, 80° 32 W). Community composition, nutrient levels, and other variables were determined in all uptake experiments. A second objective was to determine if ammonium was preferentially utilized over nitrate and to characterize further the ammonium transport system.V _max increased steadily from May until the end of July, each increase coinciding with major changes in the phytoplankton community. Cryptophyceans dominated in May, chlorophyceans in June and July, and cyanophyceans from the end of July to late October. With cyanophycean dominance,V _max declined until chlorophyceans reestablished dominance in late October. By contrast,K _m values increased from May to the end of July, but thereafter showed no correlation. Acetylene reduction experiments showed no nitrogen fixation during late summer and fall when blue-green algae were present. Preference for ammonium was implied also by negative nitrate reductase assays. Overall, the coincidence ofV _max andK _m values for [¹⁴C]-methylamine uptake and changing phytoplankton community structure suggests the possibility that successive algal communities may be changing as a result of specific species differences in ammonium affinity and uptake rates.     

The Michaelis–Menten kinetics of soil extracellular enzymes in response to temperature: a cross‐latitudinal study

Decomposition of soil organic matter (SOM) is mediated by microbial extracellular hydrolytic enzymes (EHEs). Thus, given the large amount of carbon (C) stored as SOM, it is imperative to understand how microbial EHEs will respond to global change (and warming in particular) to better predict the links between SOM and the global C cycle. Here, we measured the Michaelis–Menten kinetics [maximal rate of velocity (V_max) and half‐saturation constant (K_m)] of five hydrolytic enzymes involved in SOM degradation (cellobiohydrolase, β‐glucosidase, β‐xylosidase, α‐glucosidase, and N‐acetyl‐β‐d ‐glucosaminidase) in five sites spanning a boreal forest to a tropical rainforest. We tested the specific hypothesis that enzymes from higher latitudes would show greater temperature sensitivities than those from lower latitudes. We then used our data to parameterize a mathematical model to test the relative roles of V_max and K_m temperature sensitivities in SOM decomposition. We found that both V_max and K_m were temperature sensitive, with Q₁₀ values ranging from 1.53 to 2.27 for V_max and 0.90 to 1.57 for K_m. The Q₁₀ values for the K_m of the cellulose‐degrading enzyme β‐glucosidase showed a significant (P = 0.004) negative relationship with mean annual temperature, indicating that enzymes from cooler climates can indeed be more sensitive to temperature. Our model showed that K_m temperature sensitivity can offset SOM losses due to V_max temperature sensitivity, but the offset depends on the size of the SOM pool and the magnitude of V_max. Overall, our results suggest that there is a local adaptation of microbial EHE kinetics to temperature and that this should be taken into account when making predictions about the responses of C cycling to global change.     

Efficient synthesis of optically pure alcohols by asymmetric hydrogen-transfer biocatalysis: application of engineered enzymes in a 2-propanol–water medium

We describe an efficient method for producing both enantiomers of chiral alcohols by asymmetric hydrogen-transfer bioreduction of ketones in a 2-propanol (IPA)–water medium with E. coli biocatalysts expressing phenylacetaldehyde reductase (PAR: wild-type and mutant enzymes) from Rhodococcus sp. ST-10 and alcohol dehydrogenase from Leifsonia sp. S749 (LSADH). We also describe the detailed properties of mutant PARs, Sar268, and HAR1, which were engineered to have high activity and productivity in media composed of polar organic solvent and water, and the construction of three-dimensional structure of PAR by homology modeling. The K _m and V _max values for some substrates and the substrate specificity of mutant PARs were quite different from those of wild-type PAR. The results well explained the increased productivity of engineered PARs in IPA–water medium.     

Glucose utilization by 6pgd genotypes in Lolium perenne

To gain a deeper understanding of the mechanisms underlying associations between allozyme genotypes and rates of respiration in Lolium perenne, V_max K_m and rates of glucose flux through glycolysis and the pentose phosphate pathway were estimated for the three genotypes of the 6pgd locus. K_m V_max and V_max/K_m differed significantly among genotypes. Values of K_m for the 11, 12, and 22 genotypes were 0.29, 0.25, 0.13, while the values of V_max/K_m for die 11, 12, and 22 genotypes were 3.79, 3.85, 6.70. Flux through the pentose shunt did not differ among genotypes at 20 °C, but at 35 °C the rates of flux for the 11, 12, and 22 genotypes were 0.15, 0.25 and 0.42, respectively. Thus, the 6PGD allozyme genotypes differ markedly in both enzyme kinetic characteristics and in flux through a metabolic pathway. These associations reveal potentially causal relationships between allozyme genotypes and rates of respiration.