Enantioselective sulfation of β2-receptor agonists by the human intestine and the recombinant M-form phenolsulfotransferase

The β₂-receptor agonist class of drugs is metabolized in humans almost exclusively by sulfate conjugation. The objective of this investigation was to determine the influence of chemical structure on the stereoselectivity of the sulfoconjugation of these chiral drugs. The pure enantiomers of six β₂-agonists, including those clinically most widely used, were all effectively sulfated both by the cytosol of the human intestine and the recombinant human M-form phenolsulfotransferase (PST). Whereas the apparent K_m values (K_m,app) for the sulfation of the individual drug enantiomers by the intestinal cytosol varied widely, ranging from 4.8 μM for (S)-isoproterenol to 889 μM for (S)-albuterol, these K_m,app values were highly correlated with those obtained with M-PST (correlation coefficient 0.994). In contrast, the M-PST V_max,app values were similar for all drug enantiomers, ranging from 276 to 914 pmol min⁻¹ mg⁻¹ protein, implying that substrate binding to M-PST by far is the main determinant of the sulfation activity. For isoproterenol, the K_m,app for M-PST was 6.1 times higher for the active (R)- than for the inactive (S)-enantiomer. For other β₂-agonists, the stereoselectivity decreased towards unity as the K_m,app increased. However, for albuterol, containing a hydroxymethyl substituent at the aromatic ring, the stereoselectivity was dramatically reversed, with 10 times higher K_m,app for the inactive (S)- than for the active (R)-enantiomer. Chirality 10:800–803, 1998. © 1998 Wiley-Liss, Inc.     

Regulation of phenol sulfotransferase expression in cultured bovine bronchial epithelial cells by hydrocortisone

One conjugative pathway for the inactivation of endogenous and exogenous hydroxylated aromatic compounds is catalyzed by phenol (aryl) sulfotransferases (PSTs), which esterify phenolic acceptors with sulfate. The tracheobronchial epithelium is commonly exposed to phenolic drugs and pollutants, and metabolic sulfation and PST activity in this tissue have been previously demonstrated. To determine what factors may control PST expression, extracts of serum-free, growth factor-supplemented cultures of bovine bronchial epithelial cells were assayed for PST activity and PST antigen. The most significant finding was dose-dependent, apparent stimulated expression by hydrocortisone (EC₅₀ = 4 nM, maximal stimulation at 20 nM). Time-course experiments, however, revealed progressive loss of PST in the absence of corticosteroid. After decay of extant PST in steroid-free medium, hydrocortisone reinduced the expression of PST three to fivefold. Western blots using mouse anti-bovine PST revealed corresponding increases in 32 kDa PST protein levels in response to hydrocortisone. Steady state kinetic analyses indicated apparent K_m values of 1—3 μM for 2-naphthol regardless of culture conditions. These results suggest that detoxification of phenolic compounds by sulfation may be regulated by corticosteroids.     

Stereoselective Degradation of alpha‐Cypermethrin and Its Enantiomers in Rat Liver Microsomes

Alpha‐cypermethrin (α‐CP), [(RS)‐a‐cyano‐3‐phenoxy benzyl (1RS)‐cis‐3‐(2, 2‐dichlorovinyl)‐2, 2‐dimethylcyclopropanecarboxylate], comprises a diastereoisomer pair of cypermethrin, which are (+)‐(1R‐cis‐αS)–CP (insecticidal) and (?)‐(1S‐cis‐αR)–CP (inactive). In this experiment, the stereoselective degradation of α‐CP was investigated in rat liver microsomes by high‐performance liquid chromatography (HPLC) with a cellulose‐tris‐ (3, 5‐dimethylphenylcarbamate)‐based chiral stationary phase. The results revealed that the degradation of (?)‐(1S‐cis‐αR)‐CP was much faster than (+)‐(1R‐cis‐αS)‐CP both in enantiomer monomers and rac‐α‐CP. As for the enzyme kinetic parameters, there were some variances between rac‐α‐CP and the enantiomer monomers. In rac‐α‐CP, the V_max and CL_int of (+)‐(1R‐cis‐αS)–CP (5105.22 ± 326.26 nM/min/mg protein and 189.64 mL/min/mg protein) were about one‐half of those of (?)‐(1S‐cis‐αR)–CP (9308.57 ± 772.24 nM/min/mg protein and 352.19 mL/min/mg protein), while the K_m of the two α‐CP enantiomers were similar. However, in the enantiomer monomers of α‐CP, the V_max and K_m of (+)‐(1R‐cis‐αS) ‐CP were 2‐fold and 5‐fold of (?)‐(1S‐cis‐αR)‐CP, respectively, which showed a significant difference with rac‐α‐CP. The CL_int of (+)‐(1R‐cis‐αS)–CP (140.97 mL/min/mg protein) was still about one‐half of (?)‐(1S‐cis‐αR)–CP (325.72 mL/min/mg protein) in enantiomer monomers. The interaction of enantiomers of α‐CP in rat liver microsomes was researched and the results showed that there were different interactions between the IC₅₀ of (?)‐ to (+)‐(1R‐cis‐αS)‐CP and (+)‐ to (?)‐(1S‐cis‐αR)‐CP(IC_50(?)/(+) / IC_50(+)/(?) = 0.61). Chirality 28:58–64, 2016. © 2015 Wiley Periodicals, Inc.     

Kinetic Behaviour of Free Lipase and Mica-Based Immobilized Lipase Catalyzing the Synthesis of Sugar Esters

The utilization of natural mica as a biocatalyst support in kinetic investigations is first described in this study. The formation of lactose caprate from lactose sugar and capric acid, using free lipase (free-CRL) and lipase immobilized on nanoporous mica (NER-CRL) as a biocatalyst, was evaluated through a kinetic study. The apparent kinetic parameters, K _m and V _max, were determined by means of the Michaelis-Menten kinetic model. The Ping-Pong Bi-Bi mechanism with single substrate inhibition was adopted as it best explains the experimental findings. The kinetic results show lower K _m values with NER-CRL than with free-CRL, indicating the higher affinity of NER-CRL towards both substrates at the maximum reaction velocity (V _max,app>V _max). The kinetic parameters deduced from this model were used to simulate reaction rate data which were in close agreement with the experimental values.     

Dissecting the molecular mechanism of ion-solute cotransport: Substrate specificity mutations in theputP gene affect the kinetics of proline transport

Summary Rare mutations that alter the substrate specificity of proline permease cluster in discrete regions of theputP gene, suggesting that they may replace amino acids at the active site of the enzyme. IfputP substrate specificity mutations directly alter the active site of proline permease, the mutants should show specific defects in the kinetics of proline transport. In order to test this prediction, we examined the kinetics of threeputP substrate specificity mutants. One class of mutation increases theK _m over 120-fold but only decreases theV _max fourfold. SuchK _m mutants may be specifically defective in substrate recognition, thus identifying an amino acid critical for substrate binding. Another class of mutation decreases theV _max 80-fold without changing theK _m .V _max mutants appear to alter the rate of substrate translocation without affecting the substrate binding site. The last class of mutation alters both theK _m andV _max of proline transport. These results indicate that substrate specificity mutations alter amino acids critical for Na⁺/proline symport.     

Synthesis of (+)-(R)- and (−)-(S)-trans-8-hydroxy-2-[N-n-propyl-N-(3′-iodo-2′-propenyl)] aminotetralin: New 5-HT1A receptor ligands

(R,S)-trans-8-Hydroxy-2-[N-n-propyl-N-(3′-iodo-2′-propenyl)amino]tetralin 7 , a new radioiodinated ligand based on 8-OH-DPAT, was reported as a potential ligand for 5-HT_1A receptors. The optically active (+)-(R)- and (?)-(S)- 7 were prepared to investigate the stereoselectivity of (R,S)- 7 . Racemic intermediate 8-methoxy-2-N-n-propyltetralin was reacted with the acyl chloride of (?)-(R)-O-methylmandelic acid to form a mixture of (S,R)- and (R,R)-diastereoisomers, which were separated by flash column chromatography. After removing the N-acyl group from the diastereoisomers, the desired (+)-(R)-or (?)-(S)- 7 was obtained by adding an N-iodopropenyl group. In vitro homogenate binding studies showed the stereoselectivity of this new compound for 5-HT_1A receptors. (+)-(R)- 7 isomer displayed 100-fold higher affinity than the (?)-(S)- 7 isomer. Biochemical study indicated that (+)-(R)- 7 potently inhibited forskolin-stimulated adenylyl cyclase activity in hippocampal membranes (E_max and EC₅₀ were 24.5% and 5.4 nM, respectively), while (?)-(S)- 7 showed no effect at 1 μM. The radioiodinated (+)-(R)- and (?)-(S)-[¹²⁵I] 7 were confirmed by coelution with the resolved unlabeled compound on HPLC (reverse phase column PRP-1, acetonitrile/pH 7.0 buffer, 80/20). The active isomer, (+)-(R)-[¹²⁵I] 7 , displayed high binding affinity to 5-HT_1A receptors (K_d = 0.09 ± 0.02 nM). In contrast, the (?)-(S)- 7 isomer displayed a significantly lower affinity to the 5-HT_1A receptor (K_d > 10 nM). Thus, (+)-(R)-[¹²⁵I]trans-8-OH-PIPAT, (+)-(R)- 7 , an iodinated stereoselective 5-HT_1A receptor agonist, is potentially useful for study of in vivo and in vitro function and pharmacology of 5-HT_1A receptors in the central nervous system. © 1995 Wiley-Liss, Inc.     

In vitro inhibition of aromatase by the enantiomers of aminoglutethimide and analogs

The in vitro aromatase activity in microsomal fractions from rat ovary and its inhibition by enantiomers of aminoglutethimide (AG), rogletimide (RG), and cyclohexylaminoglutethimide (ChAG) were studied by analysing the [³H]H₂O released when [1β-³H]androstenedione was converted to estrone. Maximum velocity (V_max) and the Michaelis-Menten constant (K_m) of the microsomal aromatase enzyme were 17.40 ± 0.45 pmol/ml/mg protein/min and 1.02 ± 0.06 μM, respectively. The IC₅₀s for the enantiomers were similar for (+)-R-AG and (?)-R-ChAG (0.86 ± 0.06 and 0.89 ± 0.15 μM, respectively). (+)S-ChA'G was most potent with IC₅₀ of 0.075 ± 0.003 μM. The IC₅₀s for (?)-S-AG, (+)-R-RG, and (?)-S-RG were in the same range (23.15 ± 2.74, 24.58 ± 2.46, and 24.43 ± 2.20 μM, respectively). © 1994 Wiley-Liss, Inc.     

Expression,purification and immobilization of the intracellular invertase INVA,from <Emphasis Type="Italic">Zymomonas mobilis</Emphasis> on crystalline cellulose and Nylon-6

This paper presents two immobilization methods for the intracellular invertase (INVA), from Zymomonas mobilis. In the first method, a chimeric protein containing the invertase INVA, fused through its C-terminus to CBD _Cex from Cellulomonas fimi was expressed in Escherichia coli strain BL21 (DE3). INVA was purified and immobilized on crystalline cellulose (Avicel) by means of affinity, in a single step. No changes were detected in optimal pH and temperature when INVA-CBD was immobilized on Avicel, where values of 5.5 and 30 °C, respectively, were registered. The kinetic parameters of the INVA-CBD fusion protein were determined in both its free form and when immobilized on Avicel. K _m and V _max were affected with immobilization, since both showed an increase of up to threefold. Additionally, we found that subsequent to immobilization, the INVA-CBD fusion protein was 39% more susceptible to substrate inhibition than INVA-CBD in its free form. The second method of immobilization was achieved by the expression of a 6xHis-tagged invertase purified on Ni-NTA resin, which was then immobilized on Nylon-6 by covalent binding. An optimal pH of 5.5 and a temperature of 30 °C were maintained, subsequent to immobilization on Nylon-6 as well as with immobilization on crystalline cellulose. The kinetic parameters relating to V _max increased up to 5.7-fold, following immobilization, whereas K _m increased up to 1.7-fold. The two methods were compared showing that when invertase was immobilized on Nylon-6, its activity was 1.9 times that when immobilized on cellulose for substrate concentrations ranging from 30 to 390 mM of sucrose.     

Circadian Variation in the Uptake of Tryptophan by Cortical Synaptosomes of the Rat Brain

The kinetics of the high affinity uptake system for L-tryptophan (L-Try)have been measured over 24 hr in cortical synaptosome preparations of rat brain. Both the K_m and V_max, of the uptake process showed a statistically significant 24 hr variation. The highest K_m value, 6.71 ± 10^-5 M, was measured at the beginning of the light phase and the lowest value, 4.23 ± 10^-5 M, 6 hr into the dark phase. V_max was highest at the end of the dark phase (10.43 nmol/mg/5 min) and lowest (4.80 nmol/mg/5 min) 3 hr into the dark phase. In contrast, there was no variation over 24 hr in the V_max/K_m ratio. These results suggest that the high affinity uptake process serves to ensure a constant rate of L-tryptophan entry into the neuron in the face of circadian or ultradian variations in extracellular concentration of tryptophan.     

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.     

Crown ether activates cholesterol oxidase in low water media

Kinetic studies of cholesterol oxidase-catalysed oxidation of cholesterol in water/2-propanol mixtures showed a decrease of V _max/K _m values on the increase of concentration of the organic co-solvent. Addition of 18-crown-6 to the reaction medium results in an increase of V _max up to 16 times, and V _max/K _m up to 8.4 times, enhancing the activity of cholesterol oxidase in 2-propanol/water (88:12 v/v) to 3.5 times compared to the level observed in 46% 2-propanol.     

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.     

PARTICIPATION OF Y89 AND Y97 IN THE CONJUGATING ACTIVITY OF Drosophila melanogaster GLUTATHIONE S‐TRANSFERASE D3 (DmGSTD3)

Drosophila melanogaster glutathione S‐transferase D3 (DmGSTD3) has a shorter amino acid sequence as compared to other GSTs known in the fruit flies. This is due to the 15 amino acid N‐terminal truncation in which normally active amino acid residue is located. The work has made use of homology modeling to visualize the arrangement of amino acid side chains in the glutathione (GSH) substrate cavity. The identified amino acids were then replaced with amino acids without functional groups in the side chains and the mutants were analyzed kinetically. Homology modeling revealed that the side chains of Y89 and Y97 were shown facing toward the substrate cavity proposing their possible role in catalyzing the conjugation. Y97A and Y89A GSH gave large changes in K_m (twofold increase), V_max (fivefold reduction), and K_cat/K_m values for GSH suggesting their significant role in the conjugation reaction. The replacement at either positions has not affected the affinity of the enzyme toward 1‐chloro‐2,4‐dinitrobenzene as no significant change in values of K_max was observed. The replacement, however, had significantly reduced the catalytic efficiency of both mutants with (K_cat/K_m)^GSH and (K_cat/K_m)^CDNB of eight‐ and twofold reduction. The recombinant DmGSTD3 has shown no activity toward 1,2‐dichloro‐4‐nitrobenzene, 2,4‐hexadienal, 2,4‐heptadienal, p‐nitrobenzyl chloride, ethacrynic acid, and sulfobromophthalein. Therefore, it was evident that DmGSTD3 has made use of distal amino acids Y97 and Y89 for GSH conjugation.     

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.     

Characterization of spermine uptake by Ehrlich tumour cells in culture

Summary. Spermine is taken up by Ehrlich ascites tumour cells through a specific, saturable, temperature and energy-dependent transport system with a remarkably low affinity constant for spermine (around 1 μM). In the absence of a potassium ion gradient through the plasma membrane, spermine uptake remains saturable but the value of the K_m for spermine is much higher (153 μM). Difluormethylornithine treatment (3 mM for 48 h) induces significant increases in V_max values (up to 9-fold) and changes in the K_m values with scarce statistical significance. Among the biogenic amines tested, only spermidine and, partly, agmatine seem to share the same transport system with spermine. No difference is observed in the rate of spermine transport when assays are carried out in the presence of 50-fold excess of ornithine or calcium, or 100-fold excess of glutamine. Received April 25, 2000 Accepted November 1, 2000     

Kinetic analysis of 3H-serotonin accumulation in four regions of rat brain after lesions in the medial forebrain bundle

Kinetic analysis of ³H-serotonin accumulation by crude synaptosomal suspensions of neocortex, hippocampus and caudate or by whole homogenates of cerebellum revealed the presence of a high affinity uptake component having an apparent K_m for serotonin which ranged from 2.8 to 6.0 × 10^?8 M. A second, low affinity, uptake component with an apparent K_m of 7 × 10^?6 M was present in caudate. A comparable low affinity uptake component for serotonin was not observed in neocortex, hippocampus or cerebellum. Lesions in the medial forebrain bundle produced significant decreases in serotonin comtent of neocortes, hippocampus and caudate (66 to 75%) and a significant increase in serotonin content of cerebellum (25%). The lesions did not affect the apparent K_m of the high affinity uptake system but did produce change in V_max which paralleled the changes in content of serotonin. The lesions also produced decreases in dopamine and norepinephrine content of caudate and a comparable decrease in the V_max of the low affinity uptake system with no change in the apparent K_m. There was a correlation of 0.97 between the endogenous content of serotonin and the V_max of the high affinity uptake system. These results support the view that the high and low affinity components of serotonin uptake represent accumulation into serotonergic and catecholaminergic neurons, respectively.     

Decolorization of melanin by lignin peroxidase from<Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis>

Melanin was decolorized by lignin peroxidase fromPhanerochaete chrysosporium. This decolorization reaction showed a Michaelis-Mentens type relationship between the decolorization rate and concentration of two substrates: melanin and hydrogen peroxide. Kinetic constants of the decolorization reaction were 0.1 OD₄₇₅/min (V _max) and 99.7 mg/L (K _m) for melanin and 0.08 OD₄₇₅/min (V _max) and 504.9 μM (K _m) for hydrogen peroxide, respectively. Depletion of hydrogen peroxide interrupted the decolorization reaction, indicating the essential requirement of hydrogen peroxide. Pulsewise feeding of hydrogen peroxide continued the decolorizing reaction catalyzed by lignin peroxidase. These results indicate that enzymatic decolorization of melanin has applications in the development of new cosmetic whitening agents.     

Comparison of interactions of 5′-derivatives of deoxyoctathymidylate with human DNA polymerize α and HIV reverse transcriptase

K_m and V_max values for d(pT₈) and its derivatives containing various 5′-end groups were estimated in the reaction of DNA polymerization α catalyzed by DNA polymerase α and HIV-RT. The effect of 5′-end modification of primer is more pronounced in the case of HIV-RT. Strong influence is observed for an intercalating (ethidium) group. The affinity of EtpT₈ is 200-fold higher than that of d(pT₈). Attachment of Phn-, Dnm- and Hem-groups results in the increase of affinity of modified primer from 10 up to 20 times. For DNA polymerase α the influence of modifiers on primer affinity is much weaker. The effect of 5′-end residues on the V_max values is also more pronounced for HIV RT. The way to improve selective interaction of oligonucleotide derivatives with the primer site of HIV RT is suggested.     

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.     

Active site analysis of fructosyl amine oxidase using homology modeling and site-directed mutagenesis

A three-dimensional structural model of fructosyl amine oxidase from the marine yeast Pichia N1-1 was generated using the crystal structure of monomeric sarcosine oxidase from Bacillus sp. B-0618 as template. The putative active site region was investigated by site-directed mutagenesis, identifying several amino acid residues likely playing important roles in the enzyme reaction. Asn354 was identified as a residue that plays an important role in substrate recognition and that can be substituted in order to change substrate specificity while maintaining high catalytic activity. While the Asn354Ala substitution had no effect on the V _max K _m⁻¹ value for fructosyl valine, the V _max K _m⁻¹ value for fructosyl-^ε N-lysine was decreased 3-fold, thus resulting in a 3-fold improvement in specificity for fructosyl valine over fructosyl-^ε N-lysine.