A method of graphically analyzing substrate-inhibition kinetics

A model of substrate inhibition for enzyme catalysis was extended to describe the kinetics of photosynthetic production of ethylene by a recombinant cyanobacterium, which exhibits light-inhibition behavior similar to the substrate-inhibition behavior in enzyme reactions. To check the validity of the model against the experimental data, the model equation, which contains three kinetic parameters, was transformed so that a linear plot of the data could be made. The plot yielded reasonable linearity, and the parameter values could be estimated from the plot. The linear-plot approach was then applied to other inhibition kinetics including substrate inhibition of enzyme reactions and inhibitory growth of bacteria, whose analyses would otherwise require nonlinear least-squares fits or data measured in constrained ranges. Plots for three totally different systems all showed reasonable linearity, which enabled visual validation of the assumed kinetics. Parameter values evaluated from the plots were compared with results of nonlinear least-squares fits. A normalized linear plot for all the results discussed in this work is also presented, where dimensionless rates as a function of dimensionless concentration lie in a straight line. The linear-plot approach is expected to be complementary to nonlinear least-squares fits and other currently used methods in analyses of substrate-inhibition kinetics. Copyright 1999 John Wiley & Sons, Inc.     

Modeling and optimization of methanol as a cosolvent in Amoxicillin synthesis and its advantage over ethylene glycol

The production of semi-synthetic beta-lactam antibiotics such as Amoxicillin may be performed enzymatically using penicillin acylase under mild conditions. However, the thermodynamically favored hydrolysis of the antibiotic product and the acyl donor substrate needs to be minimized to use the kinetically controlled route. The addition of cosolvents such as ethylene glycol and methanol (the two best solvents identified so far for semi-synthetic beta-lactam antibiotics) can achieve this to some degree, but these additives also produce enzyme inhibition and deactivation. In this study, we compared ethylene glycol and methanol under various substrate conditions. Methanol gave a better synthesis to hydrolysis ratio, although its deactivating effects adversely affected production at lower cosolvent concentrations than ethylene glycol. This effect and its dependence on substrate concentration was further modeled and optimized. A few targets of optimization such as Amoxicllin level, the synthesis to hydrolysis ratio, or a combination, were employed. While maximum levels of Amoxicillin synthesis were achievable only at high substrate concentrations, improvements derived from cosolvents were most significant at low substrate concentrations.     

Kinetics of the enzymatic hydrolysis of cellulose

Enzymatic hydrolysis of cellulose for sugar production offers advantages of higher conversion, minimal by-product formation, low energy requirements, and mild operating conditions over other chemical conversions. The development of a kinetic model, based on observable, macroscopic properties of the overall system, is helpful in design and economic evaluation of processes for sugar conversion and ethanol production. A kinetic model is presented, incorporating enzyme adsorption, product inhibition, and considers a multiple enzyme and substrate system. This model was capable of simulating saccharification of a lignocellulosic material, rice straw, at high substrate (up to 333 g/L) and enzyme concentrations (up to 9.2 FPU/mL) that are common to proposed process designs.     

Methionine-induced Ethylene Production by Penicillium digitatum

Shake cultures, in contrast to static cultures of Penicillium digitatum grown in liquid medium, were induced by methionine to produce ethylene. The induction was concentration-dependent, and 7 mM was optimum for the methionine effect. In the presence of methionine, glucose (7 mM) enhanced ethylene production but did not itself induce ethylene production. The induction process lasted several hours, required the presence of viable mycelium, exhibited a lag period for ethylene production, and was effectively inhibited by cycloheximide and actinomycin D. Thus, the methionine-induced ethylene production appeared to involve induction of an enzyme system(s). Methionine not only induced ethylene production but was also utilized as a substrate since labeled ethylene was produced from [¹⁴C]methionine.     

Coleoptile removal-induced ethylene production in winter rye seedlings

Coleoptile removal-induced ethylene production was investigated in light-grown winter rye seedlings. Removal of the coleoptile induced 1-aminocyclopropane-l-carboxylic acid (ACC) synthesis and ethylene production by primary leaves and caused an inhibition of elongation growth of the leaves. The activity of ethylene-forming enzyme (EFE) was associated with the increase in ethylene evolution. Both rise in ethylene and ACC production, as well as EFE activity were inhibited by cycloheximide. Wounding the tissue 40 min after the initial treatment resulted in the second increase in ethylene evolution. Derooting of the seedlings without coleoptile removal did not induce ethylene production. It is suggested that the coleoptile represents a barrier for wound-induced ethylene production from actively growing leaf tissue.     

A comparison of the conversion of 1-amino-2-ethylcyclopropane-1-carboxylic acid stereoisomers to 1-butene by pea epicotyls and by a cell-free system

The characteristics of the conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene by pea (Pisum sativum L.) epicotyls and by pea epicotyl enzyme are compared. Of the four stereoisomers of 1-amino-2-ethylcyclopropane-1-carboxylic acid (AEC), only (1R,2S)-AEC is preferentially converted to 1-butene in pea epicotyls. This conversion is inhibited by ACC, indicating that butene production from (1R,2S)-AEC and ethylene production from ACC are catalyzed by the same enzyme. Furthermore, pea epicotyls efficiently convert ACC to ethylene with a low K _m (66 M) for ACC and do not convert 4-methylthio-2-oxo-butanoic acid (KMB) to ethylene, thus demonstrating high specificity for its substrate. In contrast, the reported pea epicotyl enzyme which catalyzes the conversion of ACC to ethylene had a high K _m (389 mM) for ACC and readily converted KMB to ethylene. We show, moreover, that the pea enzyme catalyzes the conversion of AEC isomers to butene without stereodiscrimination. Because of its lack of stereospecificity, its low affinity for ACC and its utilization of KMB as a substrate, we conclude that the reported pea enzyme system is not related to the in-vivo ethylene-forming enzyme.Abbreviations ACC 1-Amino cyclopropane-1-carboxylic acid - AEC 1-amino-2-ethylcyclopropane-1-carboxylic acid - EFE ethylene-forming enzyme - KMB 4-methylthio-2-oxobutanoic acid     

Ethylene production by growing and senescing pear fruit cell suspensions in response to gibberellin

A pear (Pyrus communis L. cv Passe Crassane) cell suspension was used as a model system to study the influence of gibberellin on processes related to fruit ripening. Growth of the cell cultures was inhibited and their loss of viability was accelerated when 0.5 millimolar gibberllic acid (GA₃) was added to suspensions at two stages of cell development, namely, growth and quiescence. Cell respiration rate was unaffected up to 2 millimolar GA₃ but ethylene production, both basal and 1-aminocyclopropane-1-carboxylic acid-induced, was inhibited at all stages of cell development. However, the degree of inhibition decreased as the cell cultures aged. The site of ethylene inhibition by GA₃ appeared to be related to the ethylene-forming enzyme. The coincident acceleration of cell senescence and inhibition of ethylene production indicate that the pear cell suspension cannot serve as an analogous model for studying the mode of action of gibberellin in delaying ripening and senescence of fruits in its entirety, although certain specific effects might be relevant.     

Biochemical basis of high-temperature inhibition of ethylene biosynthesis in ripening tomato fruits

Biggs, M. S., Woodson, W. R. and Handa, A. K. 1988. Biochemical basis of high-temperature inhibition of ethylene biosynthesis in ripening tomato fruits. Physiol. Plant. 72: 572578
Incubation of fruits of tomato ( Lycopersicon esculentum Mill. cv. Rutgers) at 34°C or above resulted in a marked decrease in ripening-associated ethylene production. High temperature inhibition of ethylene biosynthesis was not associated with permanent tissue damage, since ethylene production recovered following transfer of fruits to a permissive temperature. Determination of pericarp enzyme activities involved in ethylene biosynthesis following transfer of fruits from 25°C to 35 or 40°C revealed that 1-aminocyclopropane-l-carboxylic acid (ACC) synthase (EC 4.4.1.14) activity declined rapidly while ethylene forming enzyme (EFE) activity declined slowly. Removal of high temperature stress resulted in more rapid recovery of ACC synthase activity relative to EFE activity. Levels of ACC in pericarp tissue reflected the activity of ACC synthase before, during, and after heat stress. Recovery of ethylene production following transfer of pericarp discs from high to permissive temperature was inhibited in the presence of cycloheximide, indicating the necessity for protein synthesis. Ethylene production by wounded tomato pericarp tissue was not as inhibited by high temperature as ripening-associated ethylene production by whole fruits.     

Adenosine nucleotides and ATP synthesizing enzymes in conidia of Aspergillus niger

We have examined the effects of the enzyme inhibitors 2,4,6-trinitrobenzene sulfonic acid (TNBS) and 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) on ethylene and CO₂ production in apple and tomato fruit discs. In the past these inhibitors have been used to inhibit membrane bound enzyme systems in various animal tissues. The amino reactive inhibitor TNBS was shown to decrease ethylene production in tomato discs without affecting rates of respiration; similar results were obtained with apple. The effects of the sulfhydryl reactive inhibitor DTNB were not as clearcut as TNBS. There was little effect of DTNB on ethylene production in tomato discs, however, in apple discs ethylene production was significantly reduced. DTNB also reduced the respiration rate in apple discs, although not to the same extent as ethylene production. The inhibition of DTNB was reversed by a brief treatment with dithioerythritol. The results indicate that ethylene production takes place at the cell surface.     

Inhibition of auxin-induced ethylene production by salicylic acid in mung bean hypocotyls

Salicylic acid (SA), a common plant phenolic compound, influences diverse physiological and biochemical processes in plants. To gain insight into the mode of interaction between auxin, ethylene, and SA, the effect of SA on auxininduced ethylene production in mung bean hypocotyls was investigated. Auxin markedly induced ethylene production, while SA inhibited the auxin-induced ethylene synthesis in a dose-dependent manner. At 1 mM of SA, auxininduced ethylene production decreased more than 60% in hypocotyls. Results showed that the accumulation of ACC was not affected by SA during the entire period of auxin treatment, indicating that the inhibition of auxin-induced ethylene production by SA was not due to the decrease in ACC synthase activity, the rate-limiting step for ethylene biosynthesis. By contrast, SA effectively reduced not only the basal level of ACC oxidase activity but also the wound-and ethylene-induced ACC oxidase activity, the last step of ethylene production, in a dose-dependent manner. Northern and immuno blot analyses indicate that SA does not exert any inhibitory effect on the ACC oxidase gene expression, whereas it effectively inhibits both the in vivo and in vitro ACC oxidase enzyme activity, thereby abolishing auxin-induced ethylene production in mung bean hypocotyl tissue. It appears that SA inhibits ACC oxidase enzyme activity through the reversible interaction with Fe²⁺, an essential cofactor of this enzyme. These results are consistent with the notion that ethylene production is controlled by an intimate regulatory interaction between auxin and SA in mung bean hypocotyl tissue.     

Adsorptive bioconversion of ethylene glycol to glycolic acid by Gluconobacter oxydans DSM 2003

An integrated bioprocess for the production of glycolic acid from ethylene glycol with Gluconobacter oxydans DSM 2003 and in situ product removal were investigated. A slight substrate inhibition was observed as substrate concentration was above 20 g/l and the product inhibition was much stronger. Bioconversion of glycolic acid is an end-product-inhibited reaction. In order to increase the productivity of glycolic acid and reduce the end-product inhibition of bioconversion, an adsorptive bioconversion for glycolic acid production from ethylene glycol catalyzed by resting cells of G. oxydans DSM 2003, was developed by using anion exchange resin D315 as the adsorbent for selective removal of glycolic acid from the reaction mixture. This approach allowed the yield of glycolic acid to be increased to 93.2 g/l, compared to 74.5 g/l obtained from a conventional fed-batch mode.     

Effects of organic amines on α-aminoisobutyric acid uptake into the vacuole and on ethylene production by tomato pericarp slices

Experiments were conducted to test the possibility that organic amines inhibit ethylene production by inhibiting transport of the ethylene precursor, 1-aminocyclopro-pane-1-carboxylic acid (ACC), into the vacuole. α-Aminoisobutyric acid (αAIB) was used as a model substrate to study ACC uptake into the vacuole in relationship to ethylene production in pericarp slices of Lycopersicon esculentum Mill. cv. Liberty treated with and without organic amines and related substances. Organic amines (polyamines and other basic amines) inhibited αAIB uptake into the vacuole. These amines also enhanced ACC accumulation in the tissue and reduced the passive efflux of αAIB from the vacuole. Overall, ethylene production was inhibited. The inhibition of αAIB transport and of ethylene production followed a polyvalent cationic progression in the order polyamines > diamines> basic 1-amino acids. Ca²⁺, but not Mg²⁺, strongly stimulated αAIB uptake into the vacuole and ethylene production. At equal concentrations, Ca²⁺ counteracted the inhibitory effects of polyamines on both αAIB uptake and ethylene production. Competitive and irreversible inhibitors of polyamine biosynthesis stimulated αAIB uptake into the vacuole and ethylene production. The results indicate an apparent relationship between polyamines, ACC uptake into the vacuole and ethylene production.     

Kinetic consequences of a slow substrate binding step in group transferases. Interpretation of product inhibition experiments.

The steady state kinetic properties of a simple model for an enzyme catalyzed group transfer reaction between two substrates have been calculated. One substrate is assumed to bind slowly and the other rapidly to the enzyme. Apparent substrate inhibition or substrate activation by the rapidly binding substrate may result if the slowly binding substrate binds at unequal rates to the free enzyme and to the complex between the enzyme and the rapidly binding substrate. Competitive inhibition by each product with respect to its structurally analogous substrate is to be expected if both substrates are in rapid equilibrium with their enzyme-substrate complexes. This product inhibition pattern, however, may also be observed when one substrate binds slowly. Noncompetitive inhibition with respect to the rapidly binding substrate by its structurally analogous product may result if the slowly binding substrate binds more slowly to the enzyme-product complex than to the free enzyme. Inhibition by substrate analogs which are not products should follow the same rules as inhibition by products. Thus substrate analog inhibition experiments are not particularly informative. The form of inhibition by "transition state analog" inhibitors should reveal which substrate binds slowly. There is no sharp conceptual distinction between ordered and random "kinetic mechanisms". I therefore suggest that the use of these concepts should be abandoned.     

Steady-state kinetic mechanism of recombinant avocado ACC oxidase: initial velocity and inhibitor studies

The gaseous plant hormone ethylene modulates a wide range of biological processes, including fruit ripening. It is synthesized by the ascorbate-dependent oxidation of 1-aminocyclopropyl-1-carboxylate (ACC), a reaction catalyzed by ACC oxidase. Recombinant avocado (Persea americana) ACC oxidase was expressed in Escherichia coli and purified in milligram quantities, resulting in high levels of ACC oxidase protein and enzyme activity. An optimized assay for the purified enzyme was developed that takes into account the inherent complexities of the assay system. Fe(II) and ascorbic acid form a binary complex that is not the true substrate for the reaction and enhances the degree of ascorbic acid substrate inhibition. The K(d) value for Fe(II) (40 nM, free species) and the K(m)'s for ascorbic acid (2.1 mM), ACC (62 microM), and O(2) (4 microM) were determined. Fe(II) and ACC exhibit substrate inhibition, and a second metal binding site is suggested. Initial velocity measurements and inhibitor studies were used to resolve the kinetic mechanism through the final substrate binding step. Fe(II) binding is followed by either ascorbate or ACC binding, with ascorbate being preferred. This is followed by the ordered addition of molecular oxygen and the last substrate, leading to the formation of the catalytically competent complex. Both Fe(II) and O(2) are in thermodynamic equilibrium with their enzyme forms. The binding of a second molecule of ascorbic acid or ACC leads to significant substrate inhibition. ACC and ascorbate analogues were used to confirm the kinetic mechanism and to identify important determinants of substrate binding.     

Effect of gibberellic acid on ACC content,EFE activity and ethylene release by floral parts of the senescing carnation flower

The application of gibberellic acid via the stem of intact preclimacteric carnation flowers inhibited the climacteric surge of ethylene evolution by the flowers. Gibberellic acid also inhibited the rate of ethylene production by all individual floral parts during both the early preclimacteric (low basal level of ethylene production) and the later climacteric stages of flower development. The extent of inhibition did however, vary from one floral part to another. The most pronounced inhibition was recorded in the petal bases between the preclimacteric and senescing stages. This suggests that the petal base is an important regulatory site for ethylene production and therefore may be involved in controlling the onset and degree of petal inrolling. In all floral parts endogenous levels of ACC were reduced with GA₃ treatment, being more pronounced in the petal bases. The potential of the flowers to convert applied ACC to ethylene was not deminished by gibberellic acid.Abbreviations GA₃ gibberellic acid - ACC 1-aminocyclopropane-1-carboxylic acid - EFE ethylene forming enzyme     

Lipase-mediated hydrolysis of corn DDGS oil: Kinetics of linoleic acid production

In this study, we investigated the kinetics of linoleic acid production via lipase-mediated hydrolysis of corn DDGS oil in a batch reactor with continuous mechanical agitation and developed a kinetic model that incorporated the product inhibition to study the complete hydrolysis. The model agreed very well with observed data; though situations with low enzyme dosage or low stirring rates were modeled successfully without product inhibition, actual product concentration in such situations was too low to exert any inhibitory effects. Increasing the enzyme concentration increased hydrolysis, and beyond certain enzyme concentrations, effects tended to fade away because of excessive enzyme desorption from the interface. An enzyme dosage within the range of 40–60 KLU/L of oil dispersion could be successfully applied for a substrate concentration of 25–50 g/L of DDGS oil. Increasing the agitation rates improved enzymatic hydrolysis, but a higher stirring rate of 1000 rpm moderately improved production of linoleic acid compared with a stirring rate of 750 rpm. Within the range of substrate concentrations studied, enzymatic inhibition was moderate but still evident. The high degree of hydrolysis (i.e., ∼96% of theoretical linoleic acid yield) from DDGS oil suggests this method has potential for commercial production of linoleic acid.     

A new modeling technique and computer simulation of bacterial growth

A mathematical model that describes substrate utilization and cell growth in terms of two potentially rate-limiting enzyme systems has been developed. Consideration of substrate inhibition and enzyme repression have been incorporated. The model provides a rational approach for characterizing non-steady-state phenomena. The model has been used to analyze batch test data to illustrate the effects of inhibition, repression, and concurrent substrate utilization. Its utility lies in the fact that it provides a quantitative framework for describing changes in the activity levels of cells that result from changes in substrate concentration and/or substrate type. The lag phase resulting from exposure to a new substrate can be modeled.     

Adsorptive bioconversion of ethylene glycol to glycolic acid by Gluconobacter oxydans DSM 2003

An integrated bioprocess for the production of glycolic acid from ethylene glycol with Gluconobacter oxydans DSM 2003 and in situ product removal were investigated. A slight substrate inhibition was observed as substrate concentration was above 20 g/l and the product inhibition was much stronger. Bioconversion of glycolic acid is an end-product-inhibited reaction. In order to increase the productivity of glycolic acid and reduce the end-product inhibition of bioconversion, an adsorptive bioconversion for glycolic acid production from ethylene glycol catalyzed by resting cells of G. oxydans DSM 2003, was developed by using anion exchange resin D315 as the adsorbent for selective removal of glycolic acid from the reaction mixture. This approach allowed the yield of glycolic acid to be increased to 93.2 g/l, compared to 74.5 g/l obtained from a conventional fed-batch mode.     

Activation of aspartase by glycerol

Aspartase [EC 4.3.1.1] of Escherichia coli, which exhibits a sigmoidicity in the substrate saturation profile at alkaline pH, was markedly activated by 10–20% glycerol at low substrate concentrations and pH 8.5. In contrast, no activation, but an inhibition was observed at pH 7.0 throughout the substrate concentrations tested. The activation profile of the enzyme as a function of glycerol concentration was considerably influenced by L-aspartate concentration. Neither alteration of the cooperative nature of the enzyme nor subunit dissociation was associated with the activation. Besides glycerol, ethylene glycol, propylene glycol, dimethylsulfoxide, and dioxane also activated the enzyme.     

Ethylene formation and phenotypic analysis of transgenic tobacco plants expressing a bacterial ethylene-forming enzyme

A bacterial ethylene-forming enzyme (EFE) catalyzes oxygenation of 2-oxoglutarate to produce ethylene and carbon dioxide in contrast to a plant enzyme which uses 1-aminocyclopropane-1-carboxylic acid as a substrate. We constructed several lines of transgenic tobacco plants which expressed an EFE from Pseudomonas syringae pv. phaseolicola PK2. The gene encoding a chimeric protein consisting of EFE and beta-glucuronidase (GUS) was introduced into the tobacco genome using a binary vector which directs expression of the EFE-GUS fusion protein under the control of constitutive promoter of cauliflower mosaic virus 35S RNA. Two lines of transgenic plants produced ethylene at consistently higher rates than the untransformed plant, and their GUS activities were expressed in different tissues. A significant dwarf morphology observed in the transgenic tobacco displaying the highest ethylene production resembled the phenotype of a wild-type plant exposed to excess ethylene. These results demonstrate a potential use of bacterial EFE to supply ethylene as a hormonal signal via an alternative route using an ubiquitous substrate 2-oxoglutarate in plant tissues.