Enzyme Kinetics

We have examined the role of a salt bridge between Lys242 and Glu246 in loop L4 of procaspase 3 and of mature caspase 3, and we show that the interactions are required for stabilizing the active site. Replacing either of the residues with an alanine residue results in a complete loss of procaspase 3 activity. Although both mutants are active in the context of the mature caspase 3, the mutations result in an increase in Km and a decrease in kcat when compared with the wild-type caspase 3. In addition, the mutations result in an increase in the pKa value associated with a change in kcat with pH, but does not affect the transition observed for Km versus pH. The mutations also affect the accessibility of the active-site solvent as measured by tryptophan fluorescence emission in the presence of quenching agents and as a function of pH. We show that, as the pH is lowered, the (pro)caspase dissociates, and the mutations increase the pH-dependent instability of the dimer. Overall, the resul...

Understanding Life as regulated dissipation may help connect fields that are usually studied separately: molecular biology, bioenergetics, biophysics, bioinformatics, physiology, ecology, evolution, and nonequilibrium thermodynamics. The same principles governing enzyme nanomachines may also help explain the behavior of cells, organisms, and ecosystems. The book suggests focusing on input and output products of forces and flows in each case of free-energy transduction performed by Life. The essential novelty of Life lies in the organization that connects input and output. It is an ever-spreading power-dissipation cascade. Converting potential dissipation into internal power and an increase in external entropy is a nearly perfect bridge between Life and the universe. The book explores the possibility that biological evolution and thermodynamic evolution are coupled processes, with living systems often accelerating the dissipation of available free-energy gradients. Life thrives on dissipation. We should not neglect dissipation when describing how bioenergetics works and answering the question: "What is Life?"

The bovine pancreatic trypsin inhibitor (BPTI)-Kunitz-type protein ShPI-1 (UniProt: P31713) is the major protease inhibitor from the sea anemone Stichodactyla helianthus. This molecule is used in biotechnology and has biomedical potential related to its anti-parasitic effect. A pseudo wild-type variant, rShPI-1A, with additional residues at the Nand C-terminal, has a similar three-dimensional structure and comparable trypsin inhibition strength. Further insights into the structure-function relationship of rShPI-1A are required in order to obtain a better understanding of the mechanism of action of this sea anemone peptide. Using enzyme kinetics, we now investigated its activity against other serine proteases. Considering previous reports of bifunctional Kunitz-type proteins from anemones, we also studied the effect of rShPI-1A on voltage-gated potassium (Kv) channels. rShPI-1A binds Kv1.1, Kv1.2, and Kv1.6 channels with IC 50 values in the nM range. Hence, ShPI-1 is the first member of the sea anemone type 2 potassium channel toxins family with tight-binding potency against several proteases and different Kv1 channels. In depth sequence analysis and structural comparison of ShPI-1 with similar protease inhibitors and Kv channel toxins showed apparent non-sequence conservation for known key residues. However, we detected two subtle patterns of coordinated amino acid substitutions flanking the conserved cysteine residues at the Nand C-terminal ends.

The following experimental techniques were used to determine the structure: The reported resolution of this entry is 2.85 Å. Percentile scores (ranging between 0-100) for global validation metrics of the entry are shown in the following graphic. The table shows the number of entries on which the scores are based. Whole archive (#Entries) Similar resolution (#Entries, resolution range(Å)) R f ree 130704 3168 (2.90-2.82) Clashscore 141614 3438 (2.90-2.82) Ramachandran outliers 138981 3348 (2.90-2.82) Sidechain outliers 138945 3351 (2.90-2.82) RSRZ outliers 127900 3103 (2.90-2.82) The table below summarises the geometric issues observed across the polymeric chains and their t to the electron density. The red, orange, yellow and green segments on the lower bar indicate the fraction of residues that contain outliers for >=3, 2, 1 and 0 types of geometric quality criteria respectively. A grey segment represents the fraction of residues that are not modelled. The numeric value for each fraction is indicated below the corresponding segment, with a dot representing fractions <=5% The upper red bar (where present) indicates the fraction of residues that have poor t to the electron density. The numeric value is given above the bar. Mol Chain Length Quality of chain The following table lists non-polymeric compounds, carbohydrate monomers and non-standard residues in protein, DNA, RNA chains that are outliers for geometric or electron-density-t criteria:

Background MBLs form a large and heterogeneous group of bacterial enzymes conferring resistance to β-lactam antibiotics, including carbapenems. A large environmental reservoir of MBLs has been identified, which can act as a source for transfer into human pathogens. Therefore, structural investigation of environmental and clinically rare MBLs can give new insights into structure–activity relationships to explore the role of catalytic and second shell residues, which are under selective pressure. Objectives To investigate the structure and activity of the environmental subclass B1 MBLs MYO-1, SHD-1 and ECV-1. Methods The respective genes of these MBLs were cloned into vectors and expressed in Escherichia coli. Purified enzymes were characterized with respect to their catalytic efficiency (kcat/Km). The enzymatic activities and MICs were determined for a panel of different β-lactams, including penicillins, cephalosporins and carbapenems. Thermostability was measured and structures were...

The Met69 → Ile mutant of the OHIO-1 β-lactamase, an SHV-family enzyme, is resistant to inactivation by β-lactamase inhibitors. Analysis of purified Met69 → Ile enzyme reveals that its isoelectric point (pI 7.0) and CD spectrum are identical with those of the OHIO-1 enzyme. Levels of β-lactamase expression in Escherichia coli as determined by immunoblotting are similar for OHIO-1 and Met69 → Ile β-lactamase. The kinetic constants of the Met69 → Ile enzyme compared with OHIO-1 are smaller for benzylpenicillin (Km = 6 µM compared with 17 µM; kcat = 234 s-1 compared with 345 s-1 respectively) and carbenicillin (Km = 3 µM compared with 17 µM; kcat = 131 s-1 compared with 320 s-1 respectively). For the cephalosporins cephaloridine and 7 - (thienyl - 2 - acetamido) - 3 - [2 - (4 - N,N - dimethylaminophenylazo)pyridinium-methyl]-3-cephem-4-carboxylic acid (PADAC), a similar pattern is also seen (Km = 38 µM compared with 96 µM and 6 µM compared with 75 µM respectively; kcat = 235 s-1 compar...

L-asparaginase an enzyme belonging to the class of hydrolases which enable hydrolyze L-Asparagine to L-aspartic acid and ammonia is used in leukemia cancer treatment. L-asparaginase was purified from Enterobacter agglomerans SB 221, epiphytic bacterial symbiont of brown algae, Sargassum sp. This research aimed to optimize the concentration of L-Asn substrate, fermentation time, and CoCl2 concentration for high enzyme production. Also, the optimum pH, temperature, and CoCl2 concentration for the most efficient enzymatic activity were determined. Nessler Method determined the enzymatic activity of L-asparaginase. The optimum concentration of L-Asn substrate was found to be 10 g/L with the fermentation time of 48 hours, and the activity value of 49.572 U/mL. The optimum concentration of CoCl2 was 1.9 mM, with an activity value of 72.236 U/mL and the protein concentration of 0.2269 mg/mL. Enzyme activity was optimum at pH 8 and 37 o C temperature, yielding an activity value of 71.842 U/mL. The optimum concentration of CoCl2 was 0.25 mM, with an activity value of 111.315 U/mL. These findings suggest that the enzyme activity of L-asparaginase can be increased by the addition of 10 g/L of L-Asn as the substrate and 0.25 mM of CoCl2 as the cofactor. Sargassum binderi, the most abundant algae in the seawaters of Indonesia, can be used as a natural source of anticancer therapy, especially leukemia.

This research explores the intersection of mathematics and chemistry through the mathematical modeling of chemical reaction kinetics. By employing differential equations and numerical methods, we analyze the rates of chemical reactions and predict the concentration of reactants and products over time. The study focuses on both first-order and second-order reactions, extending to complex reaction mechanisms including reversible and consecutive reactions. Real-world chemical systems, such as enzyme kinetics and industrial catalytic process are used to validate the models. This interdisciplinary approach not only deepens the understanding of chemical behaviour but also showcases the power of mathematical tools in solving problems in the physical sciences. The paper aims to provide a framework for students and researchers to bridge the gap between theoretical mathematics and experimental chemistry.

L-Arginine deiminase from Pseudomonas aeruginosa (PaADI) catalyzes the hydrolysis of arginine to citrulline and ammonia. PaADI belongs to the guanidino group-modifying enzyme superfamily (GMSF), which conserves backbone fold and a Cys-, His-, and Asp-based catalytic core. In this paper the contributions made by the PaADI core residues Cys406, His278, and Asp166 and the contribution from the neighboring Asp280 (conserved in most but not all GMSF members) to catalysis of the formation and hydrolysis of the Cys406-alkyluronium intermediate were accessed by kinetic analysis of site-directed mutants. In addition, solution hydrolysis in a chemical model of the S-alkylthiouronium intermediate was examined to reveal the importance of general base catalysis in the enzymatic reaction. Substitutions of the active site gating residue Arg401, the L-arginine C R NH 3 + (COO -) binding residues, Arg185, Arg243, and Asn160, or the His278 hydrogen bond partner, Glu224, were found to cause dramatic reductions in the enzyme turnover rate. These results are interpreted to suggest that electrostatic interactions play a dominant role in PaADI catalysis. Structural variations observed in P. aeruginosa GMSF enzymes PaADI, agmatine deiminase (PaAgDI), and N ω ,N ω -dimethylarginine dimethylaminohydrolase (PaDDAH) indicate an early divergence of the encoding genes. Arginine analogues that are known substrates for PaAgDI and PaDDAH were tested with PaADI to define clear boundaries of biochemical function in the three hydrolases. The conservation of a catalytic core associated with the common chemical function and the divergence of substrate-binding residues (as well as one key catalytic residue) to expand the substrate range provide insight into the evolution of the catalysts that form the GMSF.

Using a multiple alignment of 175 cytochrome P450 (CYP) family 2 sequences, 20 conserved sequence motifs (CSMs) were identified with the program PCPMer. Functional importance of the CSM in CYP2B enzymes was assessed from available data on site-directed mutants and genetic variants. These analyses suggested an important role of the CSM 8, which corresponds to 187 RFDYKD 192 in CYP2B4. Further analysis showed that residues 187, 188, 190, and 192 have a very high rank order of conservation compared with 189 and 191. Therefore, eight mutants (R187A, R187K, F188A, D189A, Y190A, K191A, D192A, and a negative control K186A) were made in an N-terminal truncated and modified form of CYP2B4 with an internal mutation, which is termed 2B4dH/H226Y. Function was examined with the substrates 7-methoxy-4-(trifluoromethyl)coumarin (7-MFC), 7-ethoxy-4-(trifluoromethyl)coumarin (7-EFC), 7-benzyloxy-4-(trifluoromethyl)coumarin (7-BFC), and testosterone and with the inhibitors 4-(4-chlorophenyl)imidazole (4-CPI) and bifonazole (BIF). Compared with the template and K186A, the mutants R187A, R187K, F188A, Y190A, and D192A showed >2-fold altered substrate specificity, k cat , K m , and/or k cat /K m for 7-MFC and 7-EFC and 3-to 6-fold decreases in differential inhibition (IC 50,BIF /IC 50 , 4-CPI ). Subsequently, these mutants displayed 5-12 °C decreases in thermal stability (T m ) and 2-8 °C decreases in catalytic tolerance to temperature (T 50 ) compared with the template and K186A. Furthermore, when R187A and D192A were introduced in CYP2B1dH, the P450 expression and thermal stability were decreased. In addition, R187A showed increased activity with 7-EFC and decreased IC 50,BIF / IC 50 , 4-CPI compared with 2B1dH. Analysis of long range residue-residue interactions in the CYP2B4 crystal structures indicated strong hydrogen bonds involving Glu 149 -Asn 177 -Arg 187 -Tyr 190 and Asp 192 -Val 194 , which were significantly reduced/abolished by the Arg 187 3 Ala and Asp 192 3 Ala substitutions, respectively.

The classical Michaelis-Menten equation assumes that the Michaelis constant (Km) is independent of enzyme concentration. However, this study presents a novel Hypothesis suggesting that Km increases with enzyme concentration due to electrostatic and steric effects influencing substrate binding. By analyzing kinetic data and mathematical modeling, we demonstrate that traditional approximations such as the Quasi-Steady-State Assumption (QSSA) and Reactant Stationary Approximation (RSA) may not fully describe enzymatic behavior under varying enzyme concentrations. Our findings suggest a reevaluation in enzyme kinetics, with potential applications in metabolic regulation and drug development. The implications extend to the design of enzyme inhibitors, particularly in therapeutic strategies targeting metabolic pathways in diseases such as cancer and autoimmune disorders. This new perspective challenges certain conventional assumptions in enzymology and provides new perspectives on enzyme-substrate interactions.

Arthrobacter sialophilus neuraminidase catalyzes the hydrolysis of N-acetylneuraminyl-alpha-oxygen, nitrogen, and azido glycosides. The most effective of those substrates examined was N-acetylneuraminyl-alpha-4-methylumbelliferylglycoside (AcNeu-alpha-4-MU; Km app, 0.0193 mM; kcat, 136.4 sec-1). The products resulting from the enzymic hydrolysis of N-acetylneuraminyl-alpha-azido-glycoside were N-acetylneuraminic acid and azide ion. N-acetylneuraminyl-alpha-2,3-thiogalactylglycoside and N-acetylneuraminyl-alpha-2,6-thiogalactylglycoside were competitive inhibitors of the enzyme having KI values of 1.52 mM and 1.70 mM, respectively. Dissociation constants for these thioglycosides were also determined by fluorescence enzyme titrations which gave values similar to those determined kinetically. N-Acetylneuraminic acid, but not its methyl ester, was a competitive inhibitor of neuraminidase. Its KI value, 0.18 mM, was also determined by both methods. 5-Acetamido-2,6-anhydro-3,5-dideoxy-D-g...

Chronic fatigue syndrome (CFS) is characterized by persistent and relapsing fatigue that involves oxidative stress in its pathogenesis. We tested the hypothesis that a decrease in key carbohydratedigesting enzyme activity in the gut is one of the major biological mechanisms of developing CFS in liquid formula-fed neonatal pigs with in vivo infusion of H 2 O 2 . Piglets at 7 to 10 d of age were fi tted with an intraperitoneal catheter, allowed a 3-d post surgical recovery, and infused with either H 2 O 2 at 5 mmol/kg BW (PER; n = 8) or the same volume of saline (CON; n = 8) in six 20-ml doses daily for a period of 10 d. During this period, animal behavior was monitored, blood samples collected, and jejunal enzyme activity kinetic experiments for lactase, sucrase, maltase, and maltaseglucoamylase were conducted. Plasma concentration of reduced glutathione remained similar (P > 0.05) to the pre-infusion level over the study duration in the CON group whereas this was 65% lower (P < 0.05) than the pre-infusion level in the PER group. Piglets experiencing oxidative stress had an overall lower (P < 0.05) physical mobility and the maximal jejunal specifi c activities [μmol/(mg protein•min)] for lactase (PER, 6.54 ± 0.68 vs. CON, 12.65 ± 0.69) and maltase (PER, 57.39 ± 1.02 vs. CON, 75.60 ± 1.04), respectively. However, differences were not observed (P > 0.05) in the maximal specifi c activities [μmol/(mg protein•min)] of sucrase (PER, 10.50 ± 1.37 vs. CON, 12.40 ± 1.55) and maltase-glucoamylase (PER, 0.71 ± 0.08 vs. CON, 0.70 ± 0.07) between the 2 groups. In conclusion, infusion of a suitable dose of H 2 O 2 induced CFS in the neonatal pigs. Oxidative stress in vivo differentially affected the maximal activities of important small intestinal carbohydrate-digesting enzymes in neonatal pigs fed a dairy milk-based liquid formula.

Lopinavir (LPV) is a second-generation HIV protease inhibitor (PI) designed to overcome resistance development in patients undergoing long-term antiviral therapy. The mutation of isoleucine at position 47 of the HIV protease (PR) to alanine is associated with a high level of resistance to LPV. In this study, we show that recombinant PR containing a single I47A substitution has the inhibition constant (K i ) value for lopinavir by two orders of magnitude higher than for the wild-type PR. The addition of the I47A substitution to the background of a multiply mutated PR species from an AIDS patient showed a three-order-of-magnitude increase in K i in vitro relative to the patient PR without the I47A mutation. The crystal structure of I47A PR in complex with LPV showed the loss of van der Waals interactions in the S2/S29 subsites. This is caused by the loss of three side-chain methyl groups due to the I47A substitution and by structural changes in the A47 main chain that lead to structural changes in the flap antiparallel b-strand. Furthermore, we analyzed possible interaction of the I47A mutation with secondary mutations V32I and I54V. We show that both mutations in combination with I47A synergistically increase the relative resistance to LPV in vitro. The crystal structure of the I47A/I54V PR double mutant in complex with LPV shows that the I54V mutation leads to a compaction of the flap, and molecular modeling suggests that the introduction of the I54V mutation indirectly affects the strain of the bound inhibitor in the PR binding cleft.

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The main objective of this research was to explore the effect of the polar fraction of tubers of Pueraria tuberosa (PTWE) on DPP-IV activity. The comparison of in vitro inhibitory potential between commercially available Galvus (Vildagliptin) and PTWE was determined by measuring percent inhibition and IC50. The enzyme kinetics were also done to reveal the nature of inhibition. In vivo study was done via the glucose tolerance test and by the measurement of increased plasma GLP-1 concentration and DPP-IV activity after glucose load. 50 Conclusion: These findings suggest that antidiabetic effect of PTWE could be because of its role in DPP-IV inhibition. value of 17.4 mg/ml and was found to be a competitive inhibitor having the Ki value of 13.11 mg/ml. Plasma GLP-1 concentration was increased and DPP-IV activity was decreased after 60 min of glucose load in PTWE treated rats as compared to control rats. Overall PTWE was found to be less potential DPP-IV inhibitor than Galvus.

) inhibitory to individual cytochromes P450 (P450s) are of tremendous utility in identification of P450s responsible for the metabolism of a given drug or drug candidate in pharmaceuticals. In the present study, two inhibitory MAbs against CYP2D6 (MAb 2D6-50, IgG 2b and MAb 2D6-184 , IgG 2a ) were developed by hybridoma technology to exhibit their high specificity and potency. The MAbs were further employed to assess the quantitative role (47-93%) of CYP2D6 to the metabolism of bufuralol in human liver microsomes from seven donors. Together with the MAb inhibitory to CYP3A4 as previously reported (Mei et al., 1999), the MAbs were used to study the inhibition kinetics of dextromethorphan O-demethylation (CYP2D6), testosterone 6␤hydroxylation (CYP3A4) and aflatoxin B1 3-hydroxylation (CYP3A4), respectively, with an adequate size of sample measurement. A kinetic model was proposed to fit the experimental observations with three-dimensional nonlinear regression, thereby resulting in a solution of kinetic parameters, i.e., K I , K S , V max , ␣, and ␤ (changes in K I or K S and V max in the presence of the MAb). As a result, dissociation constants (K I ) of the MAbs for the enzymes and the maximal inhibition (␤) values for the P450-catalyzed reactions were predicted to have 0.04 to 0.25 M and >94%, respectively. The results have demonstrated that the model can accurately predict the kinetic parameters and provide some insights into the understanding of the mechanism of MAb interaction with P450 enzyme in nature and the applications of the MAbs in qualitative and quantitative identification of P450s involved in drug metabolism. Chemicals. The following chemicals were obtained from commercial sources: (Ϯ)-4-hydroxymephenytoin, (S)-mephenytoin, dextromethorphan,

) inhibitory to individual cytochromes P450 (P450s) are of tremendous utility in identification of P450s responsible for the metabolism of a given drug or drug candidate in pharmaceuticals. In the present study, two inhibitory MAbs against CYP2D6 (MAb 2D6-50, IgG 2b and MAb 2D6-184 , IgG 2a ) were developed by hybridoma technology to exhibit their high specificity and potency. The MAbs were further employed to assess the quantitative role (47-93%) of CYP2D6 to the metabolism of bufuralol in human liver microsomes from seven donors. Together with the MAb inhibitory to CYP3A4 as previously reported (Mei et al., 1999), the MAbs were used to study the inhibition kinetics of dextromethorphan O-demethylation (CYP2D6), testosterone 6␤hydroxylation (CYP3A4) and aflatoxin B1 3-hydroxylation (CYP3A4), respectively, with an adequate size of sample measurement. A kinetic model was proposed to fit the experimental observations with three-dimensional nonlinear regression, thereby resulting in a solution of kinetic parameters, i.e., K I , K S , V max , ␣, and ␤ (changes in K I or K S and V max in the presence of the MAb). As a result, dissociation constants (K I ) of the MAbs for the enzymes and the maximal inhibition (␤) values for the P450-catalyzed reactions were predicted to have 0.04 to 0.25 M and >94%, respectively. The results have demonstrated that the model can accurately predict the kinetic parameters and provide some insights into the understanding of the mechanism of MAb interaction with P450 enzyme in nature and the applications of the MAbs in qualitative and quantitative identification of P450s involved in drug metabolism. Chemicals. The following chemicals were obtained from commercial sources: (Ϯ)-4-hydroxymephenytoin, (S)-mephenytoin, dextromethorphan,

The open reading frame YLR070c of Saccharomyces cerevisiae has high sequence similarity to S. cerevisiae sorbitol dehydrogenase and to xylitol dehydrogenase of Pichia stipitis. Overexpression of this open reading frame in S. cerevisiae resulted in xylitol dehydrogenase activity. The enzyme is specific for NADH. The following Michaelis constants were estimated: D-xylulose, 1.1 mM; NADH, 240 W WM (at pH 7.0); xylitol, 25 mM; NAD, 100 W WM (at pH 9.0). Xylitol dehydrogenase activity with the same kinetic properties can also be induced by xylose in wild type S. cerevisiae cells.

Phospholipid signaling mediated by lipidderived second messengers or biologically active lipids is still new and is not well established in plants. We recently have found that lysophosphatidylethanolamine (LPE), a naturally occurring lipid, retards senescence of leaves, f lowers, and postharvest fruits. Phospholipase D (PLD) has been suggested as a key enzyme in mediating the degradation of membrane phospholipids during the early stages of plant senescence. Here we report that LPE inhibited the activity of partially purified cabbage PLD in a cell-free system in a highly specific manner. Inhibition of PLD by LPE was dosedependent and increased with the length and unsaturation of the LPE acyl chain whereas individual molecular components of LPE such as ethanolamine and free fatty acid had no effect on PLD activity. Enzyme-kinetic analysis suggested noncompetitive inhibition of PLD by LPE. In comparison, the related lysophospholipids such as lysophosphatidylcholine, lysophosphatidylglycerol, and lysophosphotidylserine had no significant effect on PLD activity whereas PLD was stimulated by lysophosphatidic acid and inhibited by lysophosphatidylinositol. Membrane-associated and soluble PLD, extracted from cabbage and castor bean leaf tissues, also was inhibited by LPE. Consistent with acyl-specific inhibition of PLD by LPE, senescence of cranberry fruits as measured by ethylene production was more effectively inhibited according to the increasing acyl chain length and unsaturation of LPE. There are no known specific inhibitors of PLD in plants and animals. We demonstrate specific inhibitory regulation of PLD by a lysophospholipid.

Phospholipid signaling mediated by lipid-derived second messengers or biologically active lipids is still new and is not well established in plants. We recently have found that lysophosphatidylethanolamine (LPE), a naturally occurring lipid, retards senescence of leaves, flowers, and postharvest fruits. Phospholipase D (PLD) has been suggested as a key enzyme in mediating the degradation of membrane phospholipids during the early stages of plant senescence. Here we report that LPE inhibited the activity of partially purified cabbage PLD in a cell-free system in a highly specific manner. Inhibition of PLD by LPE was dose-dependent and increased with the length and unsaturation of the LPE acyl chain whereas individual molecular components of LPE such as ethanolamine and free fatty acid had no effect on PLD activity. Enzyme-kinetic analysis suggested noncompetitive inhibition of PLD by LPE. In comparison, the related lysophospholipids such as lysophosphatidylcholine, lysophosphatidylgl...

Phospholipid signaling mediated by lipidderived second messengers or biologically active lipids is still new and is not well established in plants. We recently have found that lysophosphatidylethanolamine (LPE), a naturally occurring lipid, retards senescence of leaves, f lowers, and postharvest fruits. Phospholipase D (PLD) has been suggested as a key enzyme in mediating the degradation of membrane phospholipids during the early stages of plant senescence. Here we report that LPE inhibited the activity of partially purified cabbage PLD in a cell-free system in a highly specific manner. Inhibition of PLD by LPE was dosedependent and increased with the length and unsaturation of the LPE acyl chain whereas individual molecular components of LPE such as ethanolamine and free fatty acid had no effect on PLD activity. Enzyme-kinetic analysis suggested noncompetitive inhibition of PLD by LPE. In comparison, the related lysophospholipids such as lysophosphatidylcholine, lysophosphatidylglycerol, and lysophosphotidylserine had no significant effect on PLD activity whereas PLD was stimulated by lysophosphatidic acid and inhibited by lysophosphatidylinositol. Membrane-associated and soluble PLD, extracted from cabbage and castor bean leaf tissues, also was inhibited by LPE. Consistent with acyl-specific inhibition of PLD by LPE, senescence of cranberry fruits as measured by ethylene production was more effectively inhibited according to the increasing acyl chain length and unsaturation of LPE. There are no known specific inhibitors of PLD in plants and animals. We demonstrate specific inhibitory regulation of PLD by a lysophospholipid.

Thiamine (vitamin B1) deficiency due to degradation, inadequate intake, impaired absorption, or increased metabolic demand remains prevalent in both human and animal populations. In its diphosphate form (thiamine diphosphate, TDP), thiamine serves as an essential cofactor for metabolic enzymes, including transketolase (TKT), a rate-limiting enzyme of the pentosephosphate pathway. Measurement of TKT activity with and without exogenous TDP provides a functional assessment of thiamine utilization that serves as a surrogate for or complements direct quantification of thiamine forms. Beyond its historical role as a deficiency-screening tool, the TKT assay offers opportunities to interrogate enzyme function and cofactor dependence. Strategic variation of assay conditions, such as higher TDP concentrations to identify low-affinity TKT variants or including or omitting magnesium to assess functional cofactor limitation, can distinguish true deficiency from impaired enzyme utilization or altered enzyme properties. This review evaluates preanalytical variables, assay methodologies, and data presentation strategies used for TKT measurements in erythrocytes and other tissues across human and nonhuman studies. Emphasis is placed on biological and methodological determinants that shape measured activity and responsiveness. Improved interpretation frameworks and thoughtful assay design can expand the utility of TKT measurements as indicators of functional thiamine status across clinical, translational, and ecological contexts.

Dehaloperoxidase (DHP) from Amphitrite ornata is the only heme-containing, hydrogen peroxide-dependent globin capable of oxidatively dehalogenating halophenols to yield the corresponding quinones. To ascertain that this enzymatic activity is intrinsic to DHP, we have cloned and expressed the enzyme in Escherichia coli. We also find that an alternate oxygen atom donor, metachloroperbenzoic acid, gives appreciably higher activity than hydrogen peroxide. Under optimal turnover conditions (large peroxide/peracid excess), after an initial burst of activity, DHP appears to become trapped in a non-catalytic state (possibly Compound II) and is unable to fully convert all halophenol to product. However, full substrate conversion can be achieved under more physiological conditions involving a much smaller excess of oxygen atom donor. Parallel studies have been carried out using horseradish peroxidase and myoglobin to calibrate the activity of DHP versus typical peroxidase and globin proteins, respectively.

Dehaloperoxidase (DHP) from Amphitrite ornata is the only heme-containing, hydrogen peroxide-dependent globin capable of oxidatively dehalogenating halophenols to yield the corresponding quinones. To ascertain that this enzymatic activity is intrinsic to DHP, we have cloned and expressed the enzyme in Escherichia coli. We also find that an alternate oxygen atom donor, metachloroperbenzoic acid, gives appreciably higher activity than hydrogen peroxide. Under optimal turnover conditions (large peroxide/peracid excess), after an initial burst of activity, DHP appears to become trapped in a non-catalytic state (possibly Compound II) and is unable to fully convert all halophenol to product. However, full substrate conversion can be achieved under more physiological conditions involving a much smaller excess of oxygen atom donor. Parallel studies have been carried out using horseradish peroxidase and myoglobin to calibrate the activity of DHP versus typical peroxidase and globin proteins, respectively.

Dehaloperoxidase (DHP) from Amphitrite ornata is the only heme-containing, hydrogen peroxide-dependent globin capable of oxidatively dehalogenating halophenols to yield the corresponding quinones. To ascertain that this enzymatic activity is intrinsic to DHP, we have cloned and expressed the enzyme in Escherichia coli. We also find that an alternate oxygen atom donor, meta-chloroperbenzoic acid, gives appreciably higher activity than hydrogen peroxide. Under optimal turnover conditions (large peroxide/peracid excess), after an initial burst of activity, DHP appears to become trapped in a non-catalytic state (possibly Compound II) and is unable to fully convert all halophenol to product. However, full substrate conversion can be achieved under more physiological conditions involving a much smaller excess of oxygen atom donor. Parallel studies have been carried out using horseradish peroxidase and myoglobin to calibrate the activity of DHP versus typical peroxidase and globin proteins...

Kinetics of intracellular invertase production employing a double mutant derivative of Kluyveromyces marxianus was optimized by varying different process variables in a 23-litre fermentor. The maximum volumetric rate (Q P ) and invertase yield (Y P/S ) by M15 mutant were 1222 U/(L•h) and 160 U/g of substrate utilized, respectively (2-fold more than those of parental strain) at 50 °C on the molasses (150 g/L of total fermentable sugars) at pH=5.5. Glucose or sucrose (100, 150 or 170 g/L) did not repress invertase catabolically under the optimized fermentation conditions, contrary to the previous reports on other yeasts and filamentous fungi, where catabolite repression of sugars was predominant. Invertases derived by the wild (I W ) and mutant (I M ) strains were purified employing ammonium sulphate precipitation, and then characterized by column chromatographic techniques both kinetically and thermodynamically. The acidic limb of invertases was missing and collation of pK a and the heat of ionization values indicated that carboxyl groups were involved in proton transfer during active catalysis. Ratios of K cat /K m and v max /K m indicated that I M was significantly more specific for sucrose hydrolysis. The I M exhibited stability in different buffers at pH=3.0-10.0 and temperature of 50-70 °C, as reflected by long half-lives. I M showed significantly lower values of enthalpy of activation (DH*) and entropy of activation (DS*), while Gibbs free energy (DG*) was significantly increased at higher temperatures, making the I M thermodynamically more thermostable. Thus I M could be used as a catabolite-resistant invertase for the production of fructose syrup or high gravity ethanol.

The Michaelis-Menten model has and continues to be one of the most widely used models in many diverse fields. In the biomedical sciences, the model continues to be ubiquitous in biochemistry, enzyme kinetics studies, nutrition science and in the pharmaceutical sciences. Despite its wide ranging applications across disciplines, design issues for this model are given short shrift. This paper focuses on design issues and provides a variety of optimal designs of this model. In addition, we evaluate robustness properties of the optimal designs under a variation in optimality criteria. To facilitate use of optimal design ideas in practice, we design a web site for generating and comparing different types of tailor-made optimal designs and user-supplied designs for the Michaelis-Menten and related models

F420 is a microbial cofactor that mediates a wide range of physiologically important and industrially relevant redox reactions, including in methanogenesis and tetracycline biosynthesis. This deazaflavin comprises a redox-active isoalloxazine headgroup conjugated to a lactyloligoglutamyl tail. Here we studied the catalytic significance of the oligoglutamate chain, which differs in length between bacteria and archaea. We purified short-chain F420 (two glutamates) from a methanogen isolate and long-chain F420 (five to eight glutamates) from a recombinant mycobacterium, confirming their different chain lengths by HPLC and LC/MS analysis. F420 purified from both sources was catalytically compatible with purified enzymes from the three major bacterial families of F420-dependent oxidoreductases. However, long-chain F420 bound to these enzymes with a six- to ten-fold higher affinity than short-chain F420. The cofactor side chain also significantly modulated the kinetics of the enzymes, wit...

This study investigates the zone of overlap and hybridization between a new species in the Allonemobius species complex and Allonemobius socius (Scudder 1877). The new species, Allonemobius sp. nov. Tex was previously misidentiÞed as Allonemobius fasciatus (De Geer 1773). Our survey of genetic variation at 13 allozyme loci shows that the new species differs from A. fasciatus in at least two allozyme loci. A. sp. nov. Tex is distributed mainly in Texas, and it is sympatric with A. socius in some parts of its distribution where the two species hybridize. In addition to the two Þxed allelic differences between the two species, there is also a unique allele at a third locus in A. sp. nov. Tex. The proportion of hybrids in the mixed populations where the two species co-occur is always low, resulting in bimodal hybrid index scores. The bimodal distribution suggests that there is reproductive isolation between the new species and A. socius. This reproductive isolation is maintained, at least in part, through conspeciÞc sperm precedence, which also serves as the major barrier to gene ßow between A. socius and A. fasciatus.

This study investigates the zone of overlap and hybridization between a new species in the Allonemobius species complex and Allonemobius socius (Scudder 1877). The new species, Allonemobius sp. nov. Tex was previously misidentiÞed as Allonemobius fasciatus (De Geer 1773). Our survey of genetic variation at 13 allozyme loci shows that the new species differs from A. fasciatus in at least two allozyme loci. A. sp. nov. Tex is distributed mainly in Texas, and it is sympatric with A. socius in some parts of its distribution where the two species hybridize. In addition to the two Þxed allelic differences between the two species, there is also a unique allele at a third locus in A. sp. nov. Tex. The proportion of hybrids in the mixed populations where the two species co-occur is always low, resulting in bimodal hybrid index scores. The bimodal distribution suggests that there is reproductive isolation between the new species and A. socius. This reproductive isolation is maintained, at least in part, through conspeciÞc sperm precedence, which also serves as the major barrier to gene ßow between A. socius and A. fasciatus.

The investigation of specific activity of ATP sulfurylase and kinetic properties of the enzyme in cellfree extracts of intestinal bacterial strains Desulfovibrio piger Vib-7 and Desulfomicrobium sp. rod-9 is presented. the microbiological, biochemical, biophysical and statistical methods were used in the work. the optimal temperature (35ºc) and ph 8.0-8.5 for enzyme reaction were determined. An analysis of kinetic pro perties of AtP sulfurylase has been carried out. Initial (instantaneous) reaction velocity (V 0 ), maximum amount of the product of reaction (P max ), the reaction time (half saturation period, τ) and maximum velocity of the AtP sulfurylase reaction (V max ) have been defined. Michaelis constants (K m Sulfate , K m AtP , K m APS , and K m Pyrophosphate ) of the enzyme reaction were demonstrated for both D. piger Vib-7 and Desulfomicrobium sp. rod-9 intestinal bacterial strains.

Human glutathione transferases (GSTs) from Alpha (A), Mu (M) and Theta (T) classes exhibited glutathione peroxidase activity towards phospholipid hydroperoxide. The specific activities are in the order : GST A1-1 GST T1-1 GST M1-1 GST A2-2 GST A4-4. Using a specific and sensitive HPLC method, specific activities towards the phospholipid hydroperoxide, 1-palmitoyl-2-(13-hydroperoxy-cis-9,trans-11octadecadienoyl)--3-phosphatidylcholine (PLPC-OOH) were determined to be in the range of 0.8-20 nmol\min per mg of protein. Two human class Pi (P) enzymes (GST P1-1 with Ile or Val at position 105) displayed no activity towards the phospholipid hydroperoxide. Michaelis-Menten kinetics were followed only for glutathione, whereas there was a linear dependence of rate with PLPC-OOH concentration. Unlike the selenium-

Human plasmin activity is inhibited by various penicillins in a dose-dependent manner. Ampicillin and cloxacillin produce a 500 inhibition of the globinolytic activity of plasmin at 4.5 and 5.3 mm respectively. A lower inhibitory capacity is displayed by carbenicillin. Assay of plasmin by its amidolytic activity on D- valyl-L-leucyl-L-lysine p-nitroanilide dihydrochloride showed that ampicillin at a concentration producing half-maximal inhibition converted the hyperbolic activity-substrate concentration curve into a sigmoidal curve. A similar conversion occurred in the presence of ampicillin when plasmin was assayed with an alternative chromogenic substrate, L-pyroglutamyl-glycyl-L-arginine p-nitroanilide hydrochloride. 6-Aminohexanoic acid at 7.5 /IM abolished the inhibition of plasmin induced by ampicillin. The present observations suggest that ampicillin interacts with plasmin at a regulatory site different from the active site of the enzyme. The effect of 6-aminohexanoic acid indicates that the lysine-binding site may be part of a regulatory site. It is possible that modulation of plasmin activity by ligands plays a role in the control of fibrinolysis.

Background: Aminoglycoside O-phosphotransferases make up a large class of bacterial enzymes that is widely distributed among pathogens and confer a high resistance to several clinically used aminoglycoside antibiotics. Aminoglycoside 2″-phosphotransferase IVa, APH(2″)-IVa, is an important member of this class, but there is little information on the thermodynamics of aminoglycoside binding and on the nature of its rate-limiting step. Methods: We used isothermal titration calorimetry, electrostatic potential calculations, molecular dynamics simulations and X-ray crystallography to study the interactions between the enzyme and different aminoglycosides. We determined the rate-limiting step of the reaction by the means of transient kinetic measurements. Results: For the first time, K d values were determined directly for APH(2″)-IVa and different aminoglycosides. The affinity of the enzyme seems to anti-correlate with the molecular weight of the ligand, suggesting a limited degree of freedom in the binding site. The main interactions are electrostatic bonds between the positively charged amino groups of aminoglycosides and Glu or Asp residues of APH. In spite of the significantly different ratio K d /K m , there is no large difference in the transient kinetics obtained with the different aminoglycosides. We show that a product release step is rate-limiting for the overall reaction. Conclusions: APH(2″)-IVa has a higher affinity for aminoglycosides carrying an amino group in 2′ and 6′, but tighter bindings do not correlate with higher catalytic efficiencies. As with APH(3′)-IIIa, an intermediate containing product is preponderant during the steady state. General significance: This intermediate may constitute a good target for future drug design.

Systems biology approaches, such as kinetic modelling, could provide valuable insights into how thioredoxins, glutaredoxins and peroxiredoxins (here collectively called redoxins), and the systems that reduce these molecules are regulated. However, it is not clear whether redoxins should be described as redox couples (with redox potentials) or as enzymes (with Michaelis-Menten parameters) in such approaches. We show that in complete redoxin systems, redoxin substrate saturation and other purported enzymatic behaviours result from limitations in the redoxin redox cycles in these systems. Michaelis-Menten parameters are therefore inappropriate descriptors of redoxin activity; data from redoxin kinetic experiments should rather be interpreted in terms of the complete system of reactions under study. These findings were confirmed by fitting kinetic models of the thioredoxin and glutaredoxin systems to in vitro datasets. This systems approach clarifies the inconsistencies with the descriptions of redoxins and emphasizes the roles of redoxin systems in redox regulation.

High concentrations of ammonia nitrogen and low electron transfer rates lead to reduced methane production efficiency during the anaerobically digested pig slurry. The present work evaluated the impact of adding hematite-modified attapulgite on the performance of anaerobically digested pig slurry. The results indicated that the addition of hematite-modified attapulgite significantly decreased methane yield by 48.1–67.9 % at loadings of 3–10 g/L, and it also notably reduced the maximum rate of methane production. This reduction is primarily attributed to the enhanced dissimilatory reduction of hematite occurring on the surface of the attapulgite, which was supported by an increase in Fe2 + concentration and a rise in the abundance of iron-reducing bacteria (specifically, the order Burkholderiales and the phyla Chloroflexi and Acidobacteria). Consequently, this process directly and indirectly inhibited methane production and diminished the activity of methane-producing microorganisms. Furthermore, the addition of hematite-modified attapulgite decreased protease and F420 activities, adversely affecting methane production. Additionally, hematite-modified attapulgite addition altered the microbial community structure within anaerobic digestion systems and promoted a syntrophic interaction between Synergistales and hydrogenotrophic methanogens, facilitating acetate oxidation and methane production.

By using a mathematical model that predicts transport of mass and how enzymes act on molecules (i.e., enzyme kinetics), the author has demonstrated theoretically how it may be possible to use nanoscale carriers for repairing RNA damage. The primary rate-limiting factor affecting the repair of RNA is believed to be the rate at which the damaged RNA escapes from an endosome. An estimated less than 20% of the damaged RNA that is able to escape from the endosome will be effective as a therapeutic agent to repair damaged RNA. In order for the successful repair of damaged RNA to occur, there must be a sufficient concentration of cytosolic enzymes present above some threshold level, meaning many damaged RNA must be generated and repaired, then only after all of the repaired transcript dominates the cell. The study findings suggest that maximizing the efficiency of the delivery of the repairing agent and also maximizing the catalytic turnover for repairing damaged RNA would lead to improved effectiveness of repairing damaged RNA by using cytosolic enzymes to provide for greater spatiotemporal targeting and collective repair across heterogeneous tissues through a coordinated system of delivery via computer-controlled nanoscale carriers. The application of biological mathematics model to assist in the development of new therapies based on the principles of genetic engineering will aid in the shift from treating symptoms to accurately restoring the original molecular function.

The aminoacylation kinetics of T7 transcripts representing defined regions of Escherichia coli serine tRNAs were determined using purified E.coli seryl-tRNA synthetase (SerRS) and the kinetic values were used to estimate the relative contribution of various tRNAd omains to recognition by SerRS. The analysis revealed that the extra stem/loop structure, characteristic of type II tRNAs such as tRNA Ser , is the domain which makes the largest contribution to kca t /K m of aminoacylation. Moreover, K m of aminoacylation was increased by a factor of about 1000 when the extra stem/loop was changed to the consensus sequence of type I tRNA extra loops indicating that the stem structure contributes significantly to the binding of tRNA Ser to SerRS. A model RNA, which represents only the tRNA Ser coaxial acceptor-T^C stem/loop domain, was also specifically aminoacylated by SerRS having a kcat/Km about 1000-fold greater than background levels. A significant portion of the contribution of this domain to aminoacylation is attributable to the acceptor stem sequence making the acceptor stem the second most important domain for recognition by SerRS. Finally, kcat/ K m was essentially unchanged when the entire anticodon stem/loop of tRNA Ser was deleted indicating that neither the anticodon nucleotides nor the surrounding stem/loop structure are important for recognition by SerRS.

A new data treatment is described for designing kinetic experiments and analysing kinetic results for multi-substrate enzymes. Normalized velocities are plotted against normalized substrate concentrations. Data are grouped into n + 1 families across the range of substrate or product tested, n being the number of substrates plus products assayed. It has the following advantages over traditional methods: (1) it reduces to less than a half the amount of data necessary for a proper description of the system; (2) it introduces a self-consistency checking parameter that ensures the &#39;scientific reliability&#39; of the mathematical output; (3) it eliminates the need for a prior knowledge of Vmax; (4) the normalization of data allows the use of robust and fuzzy methods suitable for managing really &#39;noisy&#39; data; (5) it is appropriate for analysing complex systems, as the complete general equation is used, and the actual influence of effectors can be typified; (6) it is amenable to...

In the present research work novel ephedrine based thiourea derivative, 3-benzothioyl-1-(3-hydroxy-3-phenyl -3-propyl)-1-methylthiourea 4is synthesized and then characterized elemental analyzed via various techniques i.e., Proton NMR, carbon13 NMR and fatherly confirmed via X-ray crystallography. Compound 4 was then screened for their possible antioxidant and cytotoxic potentials. Benzoyl chloride was treated with an equimolar potassium thiocyanate in acetone to achieve benzoyl isothiocyantes. It was then treated with an equimolar (1R, 2S)-(-)-Ephedrine to obtain the 3-benzothioyl-1-(3-hydroxy-3-phenyl-3-propyl)-1-methyl thiourea4. It was then screened for antioxidant and cytotoxic potentials. The compound 4 showed excellent antioxidant activity almost comparable to ascorbic acid (standard) and have significant cytotoxic activity with LC50 value 05±0.58 ppm.

SummaryIn this article, we present a new method for optimizing designs of experiments for non‐linear mixed effects models, where a categorical factor with covariate information is a design variable combined with another design factor. The work is motivated by the need to efficiently design preclinical experiments in enzyme kinetics for a set of Human Liver Microsomes. However, the results are general and can be applied to other experimental situations where the variation in the response due to a categorical factor can be partially accounted for by a covariate. The covariate included in the model explains some systematic variability in a random model parameter. This approach allows better understanding of the population variation as well as estimation of the model parameters with higher precision.

Fedorov P., Grabic R., Fedorova G., Cosson J., Boryshpolets S., Dzyuba B. (2017): Development and application of LC/HRPS for quantification of adenine nucleotides, creatine phosphate, and creatine in sturgeon spermatozoa. Czech J. Anim. Sci., 62, 67–74. The objective of this study was to investigate the applicability of liquid chromatography coupled with high resolution product scan (LC/HRPS) mass spectrometry for simultaneous quantification of adenine nucleotides, creatine phosphate, and creatine levels after fixation of sturgeon sperm by perchloric acid. This approach has been used for the determination of adenine nucleotides, creatine, and creatine phosphate in biological tissues, but no applications to sperm cells have been reported to date. The results of method validation showed that specific correction factors for the calculation of macroergic phosphate content in sperm cells extracted by perchloric acid should be used to get accurate concentration values. The proposed LC/HRP...

The x-ray absorption edge spectra of the Cu and Fe-centers in oxidized and reduced cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase: EC 1.9.3.1) have been obtained using synchrotron radiation from the SPEAR storage ring at the Stanford Linear Accelerator Center. In addition, oxidized and reduced plastocyanin as well as a number of model copper compounds in various oxidation states were also examined. A comparison of the absorption edge fine structure of cytochrome oxidase with those of the models indicates that one of the two coopers in the oxidized protein is in the +1 oxidation state. Upon reduction of the protein with dithionite, the second copper becomes Cu(I). The shift in the Fe K-edge of cytochrome oxidase upon reduction is small (about 2 e V or 3 times 10(-19 J) and is comparable to that previously observed for the reduction of the heme iron of cytochrome c.

Two yeast manganese superoxide dismutases (MnSOD), one from Saccharomyces cerevisiae mitochondria (ScMnSOD) and the other from Candida albicans cytosol (CaMnSODc), have most biochemical and biophysical properties in common, yet ScMnSOD is a tetramer and CaMnSODc is a dimer or “loose tetramer” in solution. Although CaMnSODc was found to crystallize as a tetramer, there is no indication from the solution properties that the functionality of CaMnSODc in vivo depends upon the formation of the tetrameric structure. To elucidate further the functional significance of MnSOD quaternary structure, wild-type and mutant forms of ScMnSOD (K182R, A183P mutant) and CaMnSODc (K184R, L185P mutant) with the substitutions at dimer interfaces were analyzed with respect to their oligomeric states and resistance to pH, heat, and denaturant. Dimeric CaMnSODc was found to be significantly more subject to thermal or denaturant-induced unfolding than tetrameric ScMnSOD. The residue substitutions at dimer in...

Kinetic proofreading mechanisms explain the extraordinary accuracy observed in central biological events in terms of the enhanced specificity of substrate selection networks under a nonequilibrium environment. The nonequilibrium steady state theory incorporated with a chemical thermodynamic framework is implemented to execute a systematic investigation of dynamic and thermodynamic features of the proofreading network under continuous fuel consumption. We have identified that the dissipation-error trade-off domain of the network has a one-to-one correspondence with the deeper portion of the basin-like error rate profile depicted here. Further, quantifying the energy cost through concentration control of the chemical fuel aids in unveiling the association of the energy and chemical work with the optimal operating region of the biological error-correcting mechanism. It is shown that the proper energy content of the system, the semigrand Gibbs free energy, approaches a nominal value in the trade-off regime, whereas it gets considerably minimized in the domain with a lack of trade-off. We have also introduced a performance measuring entity corresponding to different energetic discrimination magnitudes as a product of average dissipation and coefficient of variation for the whole range of chemical fuel. Besides invoking a different perspective to the experimental and theoretical assessment of biological processes like DNA replication or protein synthesis, our findings can be advantageous in designing more efficient synthetic biological architectures. I.