Michaelis-Menten Kinetic Parameters Research Articles

Kinetics of the Dopamine Transporter in Drosophila Larva

Dopamine transporters (DAT) regulate neurotransmission and are important in diseases such as addiction and attention deficit hyperactivity disorder. The Drosophila dopamine transporter (dDAT) is analogous to the mammalian DAT, but Michaelis-Menten kinetic parameters have not been characterized in vivo. In this study, dopamine clearance kinetics were measured in a Drosophila larval CNS using an implanted carbon-fiber microelectrode and fast-scan cyclic voltammetry. Dopamine was pressure ejected from a micropipet implanted 15-20 μm from the microelectrode. Clearance of exogenously applied dopamine was significantly reduced in dDAT null (fumin) mutants, and kinetic constants in these mutants were used to determine clearance by other mechanisms including diffusion. After correction for diffusion, the maximal rate of uptake, Vmax, was estimated to be 0.11 ± 0.02 μM/s and Km was 1.3 ± 0.6 μM in wild-type flies. The clearance rate was significantly reduced following treatment with the DAT inhibitor cocaine in wild-type flies, but not in fumin mutants, which indicates that serotonin transporter is not contributing significantly to dopamine clearance in these larvae. Clearance of endogenous dopamine, evoked by optical stimulation in flies expressing Channelrhodopsin2, was similar to clearance of exogenous dopamine, but it was not possible to evoke concentrations that were close to saturation. The ability to quickly assess the role of the dopamine transporter in any Drosophila larva will be useful for future studies of how transporters regulate neurotransmission and to understand the underlying mechanisms of drug addiction.

In Vitro Metabolism and Pharmacokinetic Studies on Methylone

Abuse of the stimulant designer drug methylone (methylenedioxymethcathinone) has been documented in most parts of the world. As with many of the new designer drugs that continuously appear in the illicit drug market, little is known about the pharmacokinetics of methylone. Using in vitro studies, CYP2D6 was determined to be the primary enzyme that metabolizes methylone, with minor contributions from CYP1A2, CYP2B6, and CYP2C19. The major metabolite was identified as dihydroxymethcathinone, and the minor metabolites were N-hydroxy-methylone, nor-methylone, and dihydro-methylone. Measuring the formation of the major metabolite, biphasic Michaelis-Menten kinetic parameters were determined: V(max,1) = 0.046 ± 0.005 (S.E.) nmol/min/mg protein, K(m,1) = 19.0 ± 4.2 μM, V(max,2) = 0.22 ± 0.04 nmol/min/mg protein, and K(m,2) = 1953 ± 761 μM; the low-capacity and high-affinity contribution was assigned to the activity of CYP2D6. Additionally, a time-dependent loss of CYP2D6 activity was observed when the enzyme was preincubated with methylone, reaching a maximum rate of inactivation at high methylone concentrations, indicating that methylone is a mechanism-based inhibitor of CYP2D6. The inactivation parameters were determined to be K(I) = 15.1 ± 3.4 (S.E.) μM and k(inact) = 0.075 ± 0.005 minute(-1).

Assembly of a Concentric Förster Resonance Energy Transfer Relay on a Quantum Dot Scaffold: Characterization and Application to Multiplexed Protease Sensing

Semiconductor nanocrystals, or quantum dots (QDs), are one of the most widely utilized nanomaterials for biological applications. Their cumulative physicochemical and optical properties are both unique among nanomaterials and highly advantageous. In particular, Förster resonance energy transfer (FRET) has been widely utilized as a spectroscopic tool with QDs, whether for characterizing QD bioconjugates as a "molecular ruler" or for modulating QD luminescence "on" and "off" in biosensing configurations. Here, we investigate the assembly and utility of a new "concentric" FRET relay that comprises a central QD conjugated with multiple copies of two different peptides, each labeled with one of two fluorescent dyes, Alexa Fluor 555 (A555) or Alexa Fluor 647 (A647). Energy transfer occurs from the QD to the A555 (FRET(1)) then to the A647 (FRET(2)) and, to a lesser extent, directly from the QD to the A647 (FRET(3)). We show that such an arrangement can provide insight into the interfacial distribution of peptides assembled to the QD and can further be utilized for sensing proteolytic activity. In the latter, progress curves for digestion of the assembled peptides by two prototypical proteases, trypsin and chymotrypsin, were measured from the relative QD, A555 and A647 PL contributions, and used to extract Michaelis-Menten kinetic parameters. We further show that the concentric FRET relay, as a single nanoparticle vector, can track the tryptic activation of a proenzyme, chymotrypsinogen, to active chymotrypsin. The concentric FRET relay is thus a potentially powerful tool for the characterization of QD bioconjugates and multiplexed sensing of coupled biological activity.

Formation of enzyme polymer engineered structure for laccase and cross-linked laccase aggregates stabilization

Laccase and laccase-based cross-linked enzyme aggregates (CLEAs) were stabilized through the formation of a surrounding polymeric network made of chitosan and 3-aminopropyltriethoxysilane. The thermoresistance of the resulting enzyme polymer engineered structures of laccase (EPES-lac) and CLEAs (EPES-CLEA) were more than 30times higher than that of free laccase and CLEAs at pH 3 and 40°C. The EPES showed higher residual activity than the unmodified biocatalysts against chaotropic salts (up to 10times), EDTA (up to 5times), methanol (up to 15times) and acetone (up to 20times). The Michaelis–Menten kinetic parameters revealed that the affinity for 2,2’-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) has doubled for the EPES-lac and EPES CLEA compared to their unmodified forms. The EPES-lac structures acted optimally at pH 4 and their activity was nearly temperature-independent, while the laccase activity of EPES-CLEA was optimal at pH 4 and 60°C. Globally, the EPES have shown significantly improved properties which make them attractive candidate for the development of laccase-based applications.

Single Quantum Dot Based Nanosensor for Renin Assay

Evaluation of plasma renin activity is essential to the assessment of renin-related diseases such as hypertension, congestive heart failure, and cancers. Here, we develop a single quantum dot (QD) based nanosensor for sensitive detection of renin activity. This single-QD-based nanosensor consists of a streptavidin-coated QD and multiple biotinylated and Cy5-labeled peptide substrates, which form a QD/substrate/Cy5 complex where fluorescence resonance energy transfer (FRET) occurs with the QD as the donor and Cy5 as the acceptor. The presence of renin leads to the cleavage of the substrate and the separation of Cy5 from the QD and consequently the decrease of FRET efficiency and the reduction of Cy5 counts. Through the measurement of Cy5 counts by total internal reflection fluorescence (TIRF) microscopy, the renin activity can be quantitatively evaluated at the single-molecule level. This single-QD-based nanosensor can measure not only the renin concentration, but also the enzymatic velocity and the Michaelis-Menten kinetic parameters, and has significant advantages of simplicity, low cost with minimum sample consumption, and high sensitivity with a detection limit of 25 pM. This single-QD-based nanosensor might be further applied to monitor a variety of important enzymatic biomarkers such as kinases and endonuleases.

Steady-State Brain Glucose Concentrations During Hypoglycemia in Healthy Humans and Patients With Type 1 Diabetes

The objective of this study was to investigate the relationship between plasma and brain glucose levels during euglycemia and hypoglycemia in healthy subjects and patients with type 1 diabetes mellitus (T1DM). Hyperinsulinemic euglycemic (5 mmol/L) and hypoglycemic (3 mmol/L) [1-13C]glucose clamps were performed in eight healthy subjects and nine patients with uncomplicated T1DM (HbA1c 7.7 ± 1.4%). Brain glucose levels were measured by 13C magnetic resonance spectroscopy. Linear regression analysis was used to fit the relationship between plasma and brain glucose levels and calculate reversible Michaelis-Menten (MM) kinetic parameters. Brain glucose values during euglycemia (1.1 ± 0.4 μmol/g vs. 1.1 ± 0.3 μmol/g; P = 0.95) and hypoglycemia (0.5 ± 0.2 μmol/g vs. 0.6 ± 0.3 μmol/g; P = 0.52) were comparable between healthy subjects and T1DM patients. MM kinetic parameters of combined data were calculated to be maximum transport rate/cerebral metabolic rate of glucose (Tmax/CMRglc) = 2.25 ± 0.32 and substrate concentration at half maximal transport (Kt) = 1.53 ± 0.88 mmol/L, which is in line with previously published data obtained under hyperglycemic conditions. In conclusion, the linear MM relationship between plasma and brain glucose can be extended to low plasma glucose levels. We found no evidence that the plasma to brain glucose relationship or the kinetics describing glucose transport over the blood–brain barrier differ between healthy subjects and patients with uncomplicated, reasonably well-controlled T1DM.

A new nonpeptide substrate of human sirtuin in a capillary electrophoresis‐based assay. Investigation of the binding mode by docking experiments

Sirtuins are nicotinamide dinucleotide-dependent class III histone deacetylases catalyzing various physiological processes involved in cell proliferation, differentiation, apoptosis, and ageing. This makes them attractive targets in drug research. In order to simplify sirtuin substrates for assay development, two N(ɛ)-acetyllysine derivatives, N(ɛ)-acetyl-N(α)-(4-methyl-7-methoxycoumarin)lysine amide, and N(ɛ)-acetyl-N(α)-(4-methyl-7-methoxycoumarin)lysine methyl ester were synthesized and evaluated as substrates for human SIRT1 in a capillary electrophoresis-based enzyme assay. Substrate, deacetylated product, and the coproduct nicotinamide were separated in a 200 mM phosphate/Tris buffer at pH 2.85. Field-amplified sample injection was employed to achieve sufficient assay sensitivity. While the ester derivative was not recognized by the enzyme, the amide substrate was effectively converted to the deacetylated product. The assay was subsequently validated with respect to range, linearity, limit of detection, and limit of quantification. Michaelis-Menten kinetic parameters, K(m) = 83 μM and V(max) = 6.8 μM/min were determined. The applicability of the assay for inhibitor screening was demonstrated using the known inhibitors sirtinol and the suramin derivate NF258. Resveratrol did not increase the deacetylation rate at concentrations of up to 200 μM. Docking experiments revealed the necessity of an amide function at the C-terminus of nonpeptide substrates while more structural freedom is tolerated at the N-terminus of N(ɛ) -acetyllysine.

Rat CYP2D2, not 2D1, is functionally conserved with human CYP2D6 in endogenous morphine formation

The assumption that CYP2D1 is the corresponding rat cytochrome to human CYP2D6 has been revisited using recombinant proteins in direct enzyme assays. CYP2D1 and 2D2 were incubated with known CYP2D6 substrates, the three morphine precursors thebaine, codeine and (R)-reticuline. Mass spectrometric analysis showed that rat CYP2D2, not 2D1, catalyzed the 3-O-demethylation reaction of thebaine and codeine. In addition, CYP2D2 incubated with (R)-reticuline generated four products corytuberine, pallidine, salutaridine and isoboldine while rat CYP2D1 was completely inactive. This intramolecular phenol-coupling reaction follows the same mechanism as observed for CYP2D6. Michaelis–Menten kinetic parameters revealed high catalytic efficiencies for rat CYP2D2. These findings suggest a critical evaluation of other commonly accepted, however untested, CYP2D1 substrates.

In Vivo Tagging and Characterization of S‐Glutathionylated Proteins by a Chemoenzymatic Method

In Vivo Tagging and Characterization of S‐Glutathionylated Proteins by a Chemoenzymatic Method

Electrochemical, phosphate hydrolysis, DNA binding and DNA cleavage properties of new polyaza macrobicyclic dinickel(ii) complexes

A new class of macrobicyclic dinickel(II) complexes Ni2L1,2 B](ClO4)(4) (1-6), where L-1,L-2 are polyaza macrobicyclic binucleating ligands, and B is a N,N-donor heterocyclic base (viz. 2,2'-bipyridine (bipy) and 1,10-phenanthroline (phen)) are synthesized and characterized. The redox, catalytic, DNA binding and DNA cleavage properties were studied. They exhibit two irreversible waves in the cathodic region around E-pc = -0.95 V and E-pa = -0.85 V vs. Ag/Ag+ in CH3CN-0.1 M TBAP, respectively. The first order rate constants for the hydrolysis of 4-nitrophenylphosphate to 4-nitrophenolate by the dinickel(II) complexes 1-6 are in the range from 3.36 x 10(-5) to 10.83 x 10(-5) Ms-1. The complexes 3 and 6 show good binding propensity to calf thymus DNA giving binding constant values (K-b) in the range from 3.08 x 10(5) to 5.37 x 10(5) M-1. The binding site sizes and viscosity data suggest the DNA intercalative and/or groove binding nature of the complexes. The complexes display significant hydrolytic cleavage of supercoiled pBR322DNA at pH 7.2 and 37 degrees C. The hydrolytic cleavage of DNA by the complexes is supported by the evidence from free radical quenching and T4 ligase ligation. The pseudo Michaelis-Menten kinetic parameters k(cat) = 5.44 x 10(-2) h(-1) and K-M = 6.23 x 10(-3) M for complex 3 were obtained. Complex 3 also shows an enormous enhancement of the cleavage rate, of 1.5 x 10(6), in comparison to the uncatalysed hydrolysis rate (k = 3.6 x 10(-8) h(-1)) of ds-DNA.

Immobilization of a protease on modified chitosan beads for the depolymerization of chitosan

Abstract Neutral protease was immobilized on chitosan (CS), carboxymethyl chitosan (CMCS), and N -succinyl chitosan (NSCS) hydrogel beads. And the biocatalysts obtained were used to prepare low molecular weight chitosan (LMWC) and chitooligomers. Weight-average molecular weight of LMWC produced by neutral protease immobilized on CS, CMCS and NSCS hydrogel beads were 3.4 kDa, 3.2 kDa and 1.9 kDa, respectively. The effects of immobilization support and substrate on enzymatic reaction were analyzed by measuring classical Michaelis–Menten kinetic parameters. The FT-IR, XRD and potentiometric determination results indicated decrease of molecular weight led to transformation of crystal structure, but the degree of N -deacetylation and chemical structures of residues were not changed compared to initial chitosan. The degree of polymerization of chitooligomers was mainly from 2 to 7. We observed a strong dependence of the immobilized enzyme properties on the chemical nature of the supports, which leads to different microenvironment of neutral protease and changes the hydrolyzing process.

Low stable carbon isotope fractionation by coccolithophore RubisCO

The 13C/ 12C ratio of carbon compounds is used to identify sources and sinks in the global carbon cycle. However, the relatively enriched 13C content observed for marine organic carbon remains enigmatic. The majority of oceanic carbon is fixed by algae and cyanobacteria via the Calvin–Benson–Bassham cycle, yet isotopic discrimination by the CO 2 fixation enzyme, RubisCO (ribulose 1,5-bisphosphate carboxylase/oxygenase), has only been measured for a single marine cyanobacterium. Different forms of RubisCO occur in different phytoplankton species (overall amino acid identity varying by as much as ∼75%) and thus may vary in the degree to which they fractionate carbon. Here we measured isotope discrimination by RubisCO from the coccolithophore Emiliania huxleyi, a cosmopolitan species used as a marine algal model . E. huxleyi RubisCO discriminated substantially less ( ε = 11.1‰) against 13CO 2 than other RubisCO enzymes (18–29‰), despite having Michaelis–Menten kinetic parameters ( K CO 2 = 72 μM; V max = 0.66 μmol min −1 mg −1 protein) similar to those measured for RubisCO enzymes from different organisms. If widespread, decreased isotope discrimination of 13C by phytoplankton RubisCO may be a major factor influencing the enriched 13C content of marine organic carbon. This finding emphasizes the necessity of (a) determining ε values for RubisCOs of other marine phytoplankton and (b) re-evaluation of δ 13C values from physiological, environmental, and geological studies.

Expression and Characterization of a Novel Lipase from Aspergillus fumigatus with High Specific Activity

A novel lipase gene from Aspergillus fumigatus, afl1-1, was cloned and expressed with a molecular mass of 38 kDa in Escherichia coli for the first time. The recombinant lipase had a preference for short carbon chain p-nitrophenyl esters, especially toward C2 p-nitrophenyl ester and exhibited potent hydrolysis activity that had not been observed. The optimum pH and temperature of this new enzyme were 8.5 and 65 °C, respectively. The recombinant lipase (AFL1-1) is an alkaline enzyme which was stable in the pH range 6.0∼8.5 for 16 h (at 4 °C) and at 30∼50 °C for 1 h. It is an intracellular enzyme which was purified approximately 8.47-fold with an overall yield of 86.1% by single-step Ni-NTA affinity purification, with a very high specific activity of approximately 1.00 × 10(3) U mg(-1) on a standard substrate of p-nitrophenyl acetate. The Michaelis-Menten kinetic parameters V (max) and K (m) of the lipase were 1.37 mM mg(-1) min(-1) and 14.0 mM, respectively. Ca(2+) and other metal ions could not activate the lipase. According to the homology analysis and site-directed mutagenesis assay, the catalytic triad of the recombinant lipase was identified as Ser-165, Asp-260, and His-290 residues.

A dual enzyme electrochemical assay for the detection of organophosphorus compounds using organophosphorus hydrolase and horseradish peroxidase

Neurotoxic organophosphorus (OP) compounds are commonly used as chemical warfare agents and pesticides. Due to their high toxicity, rapid and sensitive field detection of these compounds has been an ongoing topic of interest. Biosensors made with organophosphate hydrolase enzyme (OPH) are generally designed to either amperometrically detect an electroactive leaving group produced following enzymatic cleavage, or to potenitometrically detect the pH change that occurs during cleavage. Since OPs are more likely to have phenolic leaving groups as compared to electroactive leaving groups, we have developed a new amperometric dual enzyme electrochemical assay that enables the detection of a broad class of OP compounds using the OPH enzyme combined with horseradish peroxidase (HRP). The assay has been applied to the detection of dichlofenthion, which does not have an electroactive leaving group and is not a commonly investigated OPH substrate. Using reverse phase HPLC, we have determined the Michaelis–Menten kinetic parameters of an engineered OPH enzyme to be K M = 0.11 ± 0.02 mM and k cat = 0.046 ± 0.003 s −1 with dichlofenthion as the substrate. Detection of the phenolic leaving group from the OPH enzyme reaction using the HRP electrode is carried out at −50 mV vs. Ag/AgCl where the noise and background are low and interferences are negligible. After optimization of the solution pH, the dual enzyme biosensor was found to have a limit of detection (LOD) of 24 μM (7.6 ppm), and a sensitivity of 0.095 ± 0.024 nA/μM for dichlorofenthion. By detecting the phenolic leaving groups from the OP targets using the HRP electrode, biosensors made using this new platform have the potential to detect a broad range of important OP compounds.

Phosphatidic Acid Binds and Stimulates Arabidopsis Sphingosine Kinases

Phosphatidic acid (PA) and phytosphingosine-1-phosphate (phyto-S1P) have both been identified as lipid messengers mediating plant response to abscisic acid (ABA). To determine the relationship of these messengers, we investigated the direct interaction of PA with Arabidopsis sphingosine kinases (SPHKs) that phosphorylate phytosphingosine to generate phyto-S1P. Two unique SPHK cDNAs were cloned from the annotated At4g21540 locus of Arabidopsis, and the two transcripts are differentially expressed in Arabidopsis tissues. Both SPHKs are catalytically active, phosphorylating various long-chain sphingoid bases (LCBs) and are associated with the tonoplast. They both interact with PA as demonstrated by lipid-filter binding, liposome binding, and surface plasmon resonance (SPR). SPHK1 and SPHK2 exhibited strong binding to 18:1/18:1, 16:0/18:1, and 16:0/18:2 PA, but poor binding to 16:0/16:0, 8:0/8:0, 18:0/18:0, and 18:2/18:2 PA. Surface dilution kinetics analysis indicates that PA stimulates SPHK activity by increasing the specificity constant through decreasing K(m)(B). The results show that the annotated At4g21540 locus is actually comprised of two separate SPHK genes. PA binds to both SPHKs, and the interaction promotes lipid substrate binding to the catalytic site of the enzyme. The PA-SPHK interaction depends on the PA molecular species. The data suggest that these two Arabidopsis SPHKs are molecular targets of PA, and the PA stimulation of SPHK is part of the signaling networks in Arabidopsis.

Human Vitamin K 2,3-Epoxide Reductase Complex Subunit 1-like 1 (VKORC1L1) Mediates Vitamin K-dependent Intracellular Antioxidant Function

Human vitamin K 2,3-epoxide reductase complex subunit 1-like 1 (VKORC1L1), expressed in HEK 293T cells and localized exclusively to membranes of the endoplasmic reticulum, was found to support both vitamin K 2,3-epoxide reductase (VKOR) and vitamin K reductase enzymatic activities. Michaelis-Menten kinetic parameters for dithiothreitol-driven VKOR activity were: K(m) (μM) = 4.15 (vitamin K(1) epoxide) and 11.24 (vitamin K(2) epoxide); V(max) (nmol·mg(-1)·hr(-1)) = 2.57 (vitamin K(1) epoxide) and 13.46 (vitamin K(2) epoxide). Oxidative stress induced by H(2)O(2) applied to cultured cells up-regulated VKORC1L1 expression and VKOR activity. Cell viability under conditions of no induced oxidative stress was increased by the presence of vitamins K(1) and K(2) but not ubinquinone-10 and was specifically dependent on VKORC1L1 expression. Intracellular reactive oxygen species levels in cells treated with 2,3-dimethoxy-1,4-naphthoquinone were mitigated in a VKORC1L1 expression-dependent manner. Intracellular oxidative damage to membrane intrinsic proteins was inversely dependent on VKORC1L1 expression and the presence of vitamin K(1). Taken together, our results suggest that VKORC1L1 is responsible for driving vitamin K-mediated intracellular antioxidation pathways critical to cell survival.

Hydrolysis of the soluble fluorescent molecule carboxyumbelliferyl-beta- d-glucuronide by E. coli beta-glucuronidase as applied in a rugged, in situ optical sensor

Techniques utilizing β-glucuronidase (GUS) activity as an indicator of Escherichia coli ( E. coli) presence use labeled glucuronides to produce optical signals. Carboxyumbelliferyl-β- d-glucuronide (CUGlcU) is a fluorescent labeled glucuronide that is soluble and highly fluorescent at natural water pHs and temperatures and, therefore, may be an ideal reagent for use in an in situ optical sensor. This paper reports for the first time the Michaelis–Menten kinetic parameters for the binding of E. coli GUS with CUGlcU as K m = 910 μM, V max = 41.0 μM min −1, V max/ K m 45.0 μmol L −1 min −1, the optimal pH as 6.5 ± 1.0, optimal temperature as 38 °C, and the Gibb's free energy of activation as 61.40 kJ mol −1. Additionally, it was found CUGlcU hydrolysis is not significantly affected by heavy solvents suggesting proton transfer and solvent addition that occur during hydrolysis are not limiting steps. Comparison studies were made with the more common fluorescent molecule methylumbelliferyl-β- d-glucuronide (MUGlcU). Experiments showed GUS preferentially binds to MUGlcU in comparison to CUGlcU. CUGlcU was also demonstrated in a prototype optical sensor for the detection of E. coli. Initial bench testing of the sensor produced detection of low concentrations of E. coli (1.00 × 10 3 CFU/100 mL) in 230 ± 15.1 min and high concentrations (1.05 × 10 5 CFU/100 mL) in 8.00 ± 1.01 min.

Ionic liquid-cellulose film for enzyme immobilization

β-Galactosidase was immobilized in an ionic liquid-cellulose film that presents properties as increased flexibility and formability. A polyamine was added during the preparation of the film and their presence was detected by infrared analysis. Glutaraldehyde was used to activate the films in order to generate conditions for enzyme immobilization that preserved 60% of the initial β-galactosidase activity. The Michaelis–Menten kinetics parameters, Km and Vm, for immobilized enzyme were estimated to be 11.22mM of ONPG and 460.83μmol of ONP min−1mg−1 enzyme, respectively and no marked shifts in optimum pH and temperature were observed after immobilization. Highly efficient lactose conversion was achieved in a batch process at 7°C and 35°C and was possible to reuse the new immobilized enzyme in 16 repeated cycles, at low temperature, without any drastic decrease in enzyme activity.

Production and characterization of Acidothermus cellulolyticus endoglucanase in Pichia pastoris

The endoglucanase (E1) from Acidothermus cellulolyticus has been used extensively in cellulase research. The goal of this work was to produce high levels of this enzyme in a system that facilitates purification. A codon-optimized synthetic gene for A. cellulolyticus E1 with a C-terminal histidine tag was cloned into the genome of Pichia pastoris. Strain KM71H expressed the most enzyme, with a yield of 550 mg/L culture supernatant. The temperature optimum (80 °C) and pH optimum (5.1) of the purified enzyme agree with previously determined values for the enzyme produced in other systems. Michaelis–Menten kinetic parameters were determined, using a fluorescent substrate (methylumbelliferyl-β- d-cellobioside) at various temperatures. This thermostable enzyme can be used in future cellulosic biofuels-related research.

What is the true enzyme kinetics in the biological system? An investigation of macromolecular crowding effect upon enzyme kinetics of glucose-6-phosphate dehydrogenase

Enzyme kinetic parameters for rate equations are vital in metabolic network simulation, a major part of systems biology research efforts. Measurements of Michaelis–Menten kinetic parameters K m and K cat have been performed for enzymes glucose-6-phosphate dehydrogenase (G6P DH) under crowded conditions using molecular crowding agents bovine serum albumin (BSA) and polyethylene glycol (PEG) of 8000 Da molecular weight. An increase in K cat was observed at very low concentrations of crowding agent, and also at high crowder concentrations when the experiment was performed at 45 °C with PEG. The observed pattern in K cat for G6P DH at high crowder concentrations has been explained via modelling using excluded volume theory. An increase in rate was observed at 45 °C for G6P DH versus 30 °C; this has been modelled via the Arrhenius equation.