Single-substrate Kinetics Research Articles

A reanalysis of protein tyrosine phosphatases inhibitory studies using the unnatural substrate analogue p-nitrophenyl phosphate

The determination of inhibition mode is extremely important in the understanding of drug interactions and biological mechanisms. The data presented by Hjortness et al. in their recent papers [1,2] on the inhibition of various Protein Tyrosine Phosphatases addresses this issue in an exemplary manner, determining the mode of inhibition based on global fitting of the data to multiple models of inhibition. However, Protein Tyrosine Phosphatases are known to undergo substrate induced conformational changes, so inhibition models which are based on enzyme that adhere to Michaelis-Menten single substrate kinetics may not be appropriate for examining these interactions. To examine the appropriateness of these models, the reported raw data was examined using a recently developed template for global data fitting in Excel. Based on the sum of squared residuals this analysis demonstrates that the excel template was able to match or improve on the reported fittings and demonstrates that a better fit can be achieved with a model that takes into account p-nitrophenyl phosphate-based substrate activation. Whether the substrate activation observed with this model substrate has physiological relevance is debatable, however, it does correspond to the known conformational rearrangement these enzymes undergo when working on their larger peptide substrates.

Building a model of ammonium excretion in two species of marine zooplankton based on glutamate dehydrogenase kinetics

MEPS Marine Ecology Progress Series Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsTheme Sections MEPS 550:83-99 (2016) - DOI: https://doi.org/10.3354/meps11700 Building a model of ammonium excretion in two species of marine zooplankton based on glutamate dehydrogenase kinetics I. Fernández-Urruzola1,*, N. Osma1, M. Gómez1, S. Montesdeoca-Esponda2, T. T. Packard1 1Marine Ecophysiology Group (EOMAR), Universidad de Las Palmas de Gran Canaria, 35017, Spain 2Environmental Chemical Analysis Group, Universidad de Las Palmas de Gran Canaria, 35017, Spain *Corresponding author: ifernandez@becarios.ulpgc.es ABSTRACT: NH4+ production in 2 species of marine zooplankton was predicted from glutamate dehydrogenase (GDH) activity using general bisubstrate enzyme kinetics during a period of starvation. In addition to starvation, we studied the effect of food quality on both NH4+ excretion rates (RNH4+) and the biochemical composition of zooplankton. The mathematical function used here relies on Michaelis-Menten principles and assumes that the synthesis of NH4+ is controlled mainly by the maximum velocity of GDH, the intracellular pool of substrates and the kinetic constants. This simple model described reasonably well the shifts in RNH4+ that occurred within the time course of starvation in both groups of zooplankton. The calculated RNH4+, however, underestimated the actual RNH4+. Furthermore, we used single substrate kinetics to address the magnitude of either the allosteric inhibition or the activation by purine nucleotides. We found that allosterism is more important in healthy physiological conditions. Overall, the use of first-principles kinetics seems to provide better estimates of RNH4+ than applying a general GDH/RNH4+ ratio on the measurements of GDH; therefore, its application may be of potential interest when studying zooplankton RNH4+ in heterogeneous marine ecosystems. KEY WORDS: Glutamate dehydrogenase · NH4+ excretion · Biochemical kinetics · Brachionus plicatilis · Leptomysis lingvura Full text in pdf format PreviousNextCite this article as: Fernández-Urruzola I, Osma N, Gómez M, Montesdeoca-Esponda S, Packard TT (2016) Building a model of ammonium excretion in two species of marine zooplankton based on glutamate dehydrogenase kinetics. Mar Ecol Prog Ser 550:83-99. https://doi.org/10.3354/meps11700 Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in MEPS Vol. 550. Online publication date: May 25, 2016 Print ISSN: 0171-8630; Online ISSN: 1616-1599 Copyright © 2016 Inter-Research.

INFLUENCE OF SUBSTRATE LOADING AND DETACHMENT CONDITIONS ON A PLANE BIOFILM BEHAVIOUR

Abstract A mathematical model of a plane, steady state biofilm, with the use of a single substrate kinetics, was proposed. A set of differential equations was solved. In order to analyse the biofilm’s behaviour, a number of simulations were performed. The simulations included varying process parameters such as detachment coefficient and substrate loading. Two detachment models were taken into consideration: one describing the detachment ratio as proportional to the thickness of the biofilm, and the other one proportional to the thickness of the biofilm squared. The results provided information about substrate and live cell distribution in biofilm and the influence of certain parameters on biofilm behaviour.

Continuous mathematical models of airlift bioreactors: Families, affinity, diversity and modelling for single-substrate kinetics

Continuous mathematical models of airlift bioreactors: Families, affinity, diversity and modelling for single-substrate kinetics This paper presents a method of describing an airlift bioreactor, in which biodegradation of a carbonaceous substrate described by single-substrate kinetics takes place. Eight mathematical models based on the assumption of liquid plug flow and axial dispersion flow through the riser and the downcomer in the reactor were proposed. Additionally, the impact of degassing zone with assumed complete mixing on the obtained results was analyzed. Calculations were performed for two representative hydrodynamic regimes of reactor operation, i.e. with the presence of gas bubbles only within the riser and for complete gas circulation. The conclusions related to the apparatus design and process performance under sufficient aeration of the reaction mixture were drawn on the basis of the obtained results.

Microbial Removal of Phenol and P-Chlorophenol from Industrial Waste Water Using Rhodococcus sp.RSP8 and Its Growth Kinetic Modeling

A phenol-degrading microorganism, Rhodococcus sp.RSP8, was used to study the substrate interactions during cell growth on phenol and p-chlorophenol dual substrates. Both phenol and p-chlorophenol could be utilized by the bacteria as the sole carbon and energy sources. When cells grew on the mixture of phenol and p-chlorophenol, strong substrate interactions were observed. The p-chlorophenol inhibited the degradation of phenol, on the other hand, phenol also inhibited the utilization of p-chlorophenol. The overall cell growth rate depends on the co-actions of phenol and p-chlorophenol. In addition, the cell growth and substrate degradation kinetics of phenol, p-chlorophenol as single and mixed substrates for Rhodococcus sp.RSP8 in batch cultures were also investigated over a wide range of initial phenol concentrations (5-1600 mg.L–1) and initial p-chlorophenol concentrations (5 – 250 mg.L–1). The single-substrate kinetics was described well using the Haldane-type kinetic models, with model constants of µm1 = 0.15 h–1, KS1 = 2.22 mg.L–1 and Ki1 = 245.37 mg.L–1 for cell growth on phenol and of µm2 = 0.0782 h–1, KS2 = 1.30 mg.L–1 and Ki2 = 71.77 mg.L–1, K′i2 = 5480 (mg.L–1)2 for cell growth on p-chlorophenol. Proposed cell growth kinetic model was used to characterize the substrates interactions in the dual substrates system.

Dual substrates biodegradation kinetics of m-cresol and pyridine by Lysinibacillus cresolivorans

Phenols and N-heterocyclic compounds are found to co-exist in actual wastewater, especially in petrochemical and coking wastewater. Lysinibacillus cresolivorans, a bacterium capable of phenol-biodegradation was used to study the substrate interactions of m-cresol and pyridine as single and dual substrates. The cell growth and substrate biodegradation kinetics were also investigated with initial m-cresol concentrations varying from 0 to 1200mg/L and pyridine concentrations varying from 0 to 150mg/L. The single substrate kinetics was well described by the Haldane kinetic models. The single-substrate parameter values of m-cresol on cell growth were μmax=0.89h−1, Ks=426.25mg/L, Ki=51.26mg/L and μmax=0.0925h−1, Ks=60.28mg/L, Ki=16.17mg/L for cell growth on pyridine. Inhibitory effects of substrates were observed when cells were grown on the mixed substrates. The interaction parameter Im,p (0.76) was greater than Im,p (0.11), which indicated that m-cresol inhibited the utilization of pyridine much more than pyridine inhibited the biodegradation of m-cresol. The study showed a good potential of L. cresolivorans in degrading mixed substrates of m-cresol and pyridine.

Kinetic modeling of growth and biodegradation of phenol and m-cresol using Alcaligenes faecalis

A phenol-degrading microorganism, Alcaligenes faecalis, was used to study the substrate interactions during cell growth on phenol and m-cresol dual substrates. Both phenol and m-cresol could be utilized by the bacteria as the sole carbon and energy sources. When cells grew on the mixture of phenol and m-cresol, strong substrate interactions were observed. m-Cresol inhibited the degradation of phenol, on the other hand, phenol also inhibited the utilization of m-cresol, the overall cell growth rate was the co-action of phenol and m-cresol. In addition, the cell growth and substrate degradation kinetics of phenol, m-cresol as single and mixed substrates for A. faecalis in batch cultures were also investigated over a wide range of initial phenol concentrations (10–1400 mg L −1) and initial m-cresol concentrations (5–200 mg L −1). The single-substrate kinetics was described well using the Haldane-type kinetic models, with model constants of μ m1 = 0.15 h −1, K S1 = 2.22 mg L −1 and K i1 = 245.37 mg L −1 for cell growth on phenol and μ m2 = 0.0782 h −1, K S2 = 1.30 mg L −1 and K i2 = 71.77 mgL −1, K ′ i 2 = 5480 ( mg L − 1 ) 2 for cell growth on m-cresol. Proposed cell growth kinetic model was used to characterize the substrates interactions in the dual substrates system, the obtained parameters representing interactions between phenol and m-cresol were, K = 1.8 × 10 −6, M = 5.5 × 10 −5, Q = 6.7 × 10 −4. The results received in the experiments demonstrated that these models adequately described the dynamic behaviors of phenol and m-cresol as single and mixed substrates by the strain of A. faecalis.

Functional Profiling of Recombinant NS3 Proteases from All Four Serotypes of Dengue Virus Using Tetrapeptide and Octapeptide Substrate Libraries

Regulated proteolysis by the two-component NS2B/NS3 protease of dengue virus is essential for virus replication and the maturation of infectious virions. The functional similarity between the NS2B/NS3 proteases from the four genetically and antigenically distinct serotypes was addressed by characterizing the differences in their substrate specificity using tetrapeptide and octapeptide libraries in a positional scanning format, each containing 130,321 substrates. The proteases from different serotypes were shown to be functionally homologous based on the similarity of their substrate cleavage preferences. A strong preference for basic amino acid residues (Arg/Lys) at the P1 positions was observed, whereas the preferences for the P2-4 sites were in the order of Arg > Thr > Gln/Asn/Lys for P2, Lys > Arg > Asn for P3, and Nle > Leu > Lys > Xaa for P4. The prime site substrate specificity was for small and polar amino acids in P1' and P3'. In contrast, the P2' and P4' substrate positions showed minimal activity. The influence of the P2 and P3 amino acids on ground state binding and the P4 position for transition state stabilization was identified through single substrate kinetics with optimal and suboptimal substrate sequences. The specificities observed for dengue NS2B/NS3 have features in common with the physiological cleavage sites in the dengue polyprotein; however, all sites reveal previously unrecognized suboptimal sequences.

Fermentation kinetics of ethanol production from glucose and xylose by recombinant Saccharomyces 1400(pLNH33).

Fermentation kinetics of ethanol production from glucose, xylose, and their mixtures using a recombinant Saccharomyces 1400(pLNH33) are reported. Single-substrate kinetics indicate that the specific growth rate of the yeast and the specific ethanol productivity on glucose as the substrate was greater than on xylose as a substrate. Ethanol yields from glucose and xylose fermentation were typically 95 and 80% of the theoretical yield, respectively. The effect of ethanol inhibition is more pronounced for xylose fermentation than for glucose fermentation. Studies on glucose-xylose mixtures indicate that the recombinant yeast co-ferments glucose and xylose. Fermentation of a 52.8 g/L glucose and 56.3 g/L xylose mixture gave an ethanol concentration of 47.9 g/L after 36 h. Based on a theoretical yield of 0.51 g ethanol/g sugars, the ethanol yield from this experiment (for data up to 24 h) was calculated to be 0.46 g ethanol/g sugar or 90% of the theoretical yield. The specific growth rate of the yeast on glucose-xylose mixtures was found to lie between the specific growth rate on glucose and the specific growth rate on xylose. Kinetic studies were used to develop a fermentation model incorporating the effects of substrate inhibition, product inhibition, and inoculum size. Good agreements were obtained between model predictions and experimental data from batch fermentation of glucose, xylose, and their mixtures.

Growth kinetics of suspended microbial cells: from single-substrate-controlled growth to mixed-substrate kinetics.

Growth kinetics, i.e., the relationship between specific growth rate and the concentration of a substrate, is one of the basic tools in microbiology. However, despite more than half a century of research, many fundamental questions about the validity and application of growth kinetics as observed in the laboratory to environmental growth conditions are still unanswered. For pure cultures growing with single substrates, enormous inconsistencies exist in the growth kinetic data reported. The low quality of experimental data has so far hampered the comparison and validation of the different growth models proposed, and only recently have data collected from nutrient-controlled chemostat cultures allowed us to compare different kinetic models on a statistical basis. The problems are mainly due to (i) the analytical difficulty in measuring substrates at growth-controlling concentrations and (ii) the fact that during a kinetic experiment, particularly in batch systems, microorganisms alter their kinetic properties because of adaptation to the changing environment. For example, for Escherichia coli growing with glucose, a physiological long-term adaptation results in a change in KS for glucose from some 5 mg liter-1 to ca. 30 microg liter-1. The data suggest that a dilemma exists, namely, that either "intrinsic" KS (under substrate-controlled conditions in chemostat culture) or micromax (under substrate-excess conditions in batch culture) can be measured but both cannot be determined at the same time. The above-described conventional growth kinetics derived from single-substrate-controlled laboratory experiments have invariably been used for describing both growth and substrate utilization in ecosystems. However, in nature, microbial cells are exposed to a wide spectrum of potential substrates, many of which they utilize simultaneously (in particular carbon sources). The kinetic data available to date for growth of pure cultures in carbon-controlled continuous culture with defined mixtures of two or more carbon sources (including pollutants) clearly demonstrate that simultaneous utilization results in lowered residual steady-state concentrations of all substrates. This should result in a competitive advantage of a cell capable of mixed-substrate growth because it can grow much faster at low substrate concentrations than one would expect from single-substrate kinetics. Additionally, the relevance of the kinetic principles obtained from defined culture systems with single, mixed, or multicomponent substrates to the kinetics of pollutant degradation as it occurs in the presence of alternative carbon sources in complex environmental systems is discussed. The presented overview indicates that many of the environmentally relevant apects in growth kinetics are still waiting to be discovered, established, and exploited.

In vitro formation of retinoic acid from retinal in rat liver

Enzymatic conversion of retinal to retinoic acid in rat liver cytosol was detected using a rapid and sensitive assay based on high pressure liquid chromatography (HPLC). This retinal oxidase assay system did not require extraction steps or any other manipulation of the sample mixture once the sample vial was sealed for incubation. The product (retinoic acid) and the reactant (retinal) were separated by HPLC in 14.0 min with a sensitivity of 15 and 40 pmol per injection for retinoic acid and retinal, respectively. Enzymatic activity was observed to be linear with protein concentration (0-2.4 mg/mL) and time (0-30 min) and displayed a broad pH maximum of 7.7-9.7. The enzyme exhibited Michaelis-Menten single-substrate kinetics with an apparent Km of 0.25 mM. The average specific activity in nine normal rats was 35.6 +/- 3.3 nmol retinoic acid formed/h per mg protein. Incubation of the enzyme with zinc did not affect the rate of retinoic acid synthesis. Dithiothreitol inhibited the reaction. Both NAD and NADH stimulated retinoic acid formation. Formation of retinol was also observed when these pyridine nucleotides were added to the reaction mixture, indicating the presence of retinal reductase activity. The results of kinetic studies suggest that NADH may act indirectly to stimulate retinoic acid formation.

The catalytic bimodality of mammalian phosphoglycerate mutase.

Rabbit muscle phosphoglycerate mutase, presumed to manifest an absolute cofactor requirement for activity, has been found to express catalysis (3 +/- 1% of optimum) in the absence of added D-glycerate-2,3-P2. Isotope experiments indicate that this catalysis proceeds through a binary phosphoryl enzyme-glycerate intermediate which dissociates into free enzyme and monophosphoglycerate. 32P-Labeled phosphoglycerate mutase is formed by reaction with either D-32P-glycerate-3-P or D-U32P-glycerate-2,3-P2. In each case, the acid lability and alkali stability of the covalent adduct, phosphoenzyme, is consistent with a phosphohistidyl residue having been formed within the active site. D-[U-14C]Glycerate reacts with phosphoenzyme to generate D-[U-14C]monophosphoglycerate which, in turn, can react further with phosphoenzyme to yield D-[U-14C]glycerate-2,3-P2. The pH profile for the cofactor-independent activity exhibits an optimum at 6.0 as opposed to 7.0 when D-glycerate-2,3-P2 is present in the reaction medium. Bisubstrate kinetics (pH 7.0, 23 degrees C) with D-glycerate-3-P concentration as the variable, yields a family of reciprocal plots which is in accord with a modified ping-pong mechanism when D-glycerate-2,3-P2 concentrations are greater than 10(-1) Km (where Km = 0.33 microM). Progressively diminishing concentrations (much less than Km) of D-glycerate-2,3-P2 produce curvilinear reciprocal plots that approach linearity as a limit in accordance with single substrate kinetics.

Yeast alcohol dehydrogenase immobilized in a glutaraldehyde–albumin matrix: Kinetics and cofactor diffusional effects*

AbstractThis study was carried out to define how the overall rate of reaction would be influenced by different degrees of diffusional resistance to cofactor transport within an oxidoreductase membrane matrix. To accomplish this, 0.7–6.6μM yeast alcohol dehydrogenase was immobilized in an albumin matrix crosslinked with 2.5 or 5.0% glutaraldehyde to give 102–1685 μM thick membranes. The enzyme half‐life was at least doubled at pH 7.5 or 8.8 on immobilization. Values of the kinetic constants for the soluble and immobilized enzyme were determined at 25°C and pH 8.8 over the range of 0.01–1.0M bulk solution concentration of ethanol as substrate and 140–1000μM bulk solution concentration of nicotinamide adenine dinucleotide (NAD+) as cofactor, to give essentially single substrate kinetics in NAD+. Equilibrium partitioning of ethanol and NAD+ between the solution and membrane was measured and used in the data analysis. The four kinetic constants for the soluble enzyme agreed with literature values; and all increased with immobilization of the enzyme. The Michaelis constants for NAD+ and for ethanol were greater for the immobilized enzyme. The diffusional resistance to NAD+ transport, presented in terms of the Thiele modulus, showed that the overall rate of reaction was decreased by about 50% even at values of the modulus as low as 2.0.