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PubMed2001

Origin of apparent fast and non-exponential kinetics of lysozyme folding measured in pulsed hydrogen exchange experiments.

Folding of lysozyme at pH 5.2 is a complex processes. After rapid collapse (<1 ms) kinetic partitioning into a slow and fast folding pathway occurs. The fast pathway leads directly to the native structure (N), whereas the slow pathway goes through a partially folded intermediate (I(1)) with native-like secondary structure in the alpha-domain. This mechanism is in agreement with data from a large number of spectroscopic probes, from changes in the radius of gyration and from measurements on the time-course of the populations of the different species. Results from pulsed hydrogen exchange experiments, in contrast, revealed that the secondary structure of I(1) and of N is formed significantly faster than changes in spectroscopic properties occur and showed large variations in the protection kinetics of individual amide sites. We investigated the molecular origin of the rapid amide protection by quantitatively simulating all kinetic processes during the pulse-labeling experiments. Absorbance and fluorescence-detected folding kinetics showed that the early events in lysozyme folding are accelerated under exchange conditions (pH 9.2) and that a change in folding mechanism occurs due to the transient population of an additional intermediate (I(2)). This leads to kinetic competition between exchange and folding during the exchange pulse and to incomplete labeling of amide sites with slow intrinsic exchange rates. As a result, apparently faster and non-exponential kinetics of amide protection are measured in the labeling experiments. Our results further suggest that collapsed lysozyme (C) and I(1) have five and ten-times reduced free exchange rates, respectively, due to limited solvent accessibility.

Amides↗

PubMed2002

Base-sequence specificity of Hoechst 33258 and DAPI binding to five (A/T)4 DNA sites with kinetic evidence for more than one high-affinity Hoechst 33258-AATT complex.

The binding of Hoechst 33258 and DAPI to five different (A/T)4 sequences in a stable DNA hairpin was studied exploiting the substantial increase in dye fluorescence upon binding. The two dyes have comparable affinities for the AATT site (e.g. association constant K(a)=5.5 x 10(8) M(-1) for DAPI), and their affinities decrease in the series AATT >> TAAT approximately equal to ATAT > TATA approximately equal to TTAA. The extreme values of K(a) differ by a factor of 200 for Hoechst 33258 but only 30 for DAPI. The binding kinetics of Hoechst 33258 were measured by stopped-flow under pseudo-first order conditions with an (A/T)4 site in excess. The lower-resolution experiments can be well represented by single exponential processes, corresponding to a single-step binding mechanism. The calculated association-rate parameters for the five (A/T)4 sites are similar (2.46 x 10(8) M(-1) s(-1) to 0.86 x 10(8) M(-1) s(-1)) and nearly diffusion-controlled, while the dissociation-rate parameters vary from 0.42 s(-1) to 96 s(-1). Thus the association constants are kinetically controlled and are close to their equilibrium-determined values. However, when obtained with increased signal-to-noise ratio, the kinetic traces for Hoechst 33258 binding at the AATT site reveal two components. The concentration dependencies of the two time constants and amplitudes are consistent with two different kinetically equivalent two-step models. In the first model, fast bimolecular binding is followed by an isomerization of the initial complex. In the second model, two single-step associations form two complexes that mutually exclude each other. For both models the four reaction-rate parameters are calculated. Finally, specific dissociation kinetics, using poly[d(A-5BrU)], show that the kinetics are even more complex than either two-step model. We correlate our results with the different binding orientations and locations of Hoechst 33258 in the DNA minor groove found in several structural studies in the literature.

AT Rich Sequence↗

PubMed1999

Kinetic models and phenomenological analysis of passive lipid translocation in single-file.

Passive movement of lipids through a membrane-embedded pore is analysed with kinetic equations of transport in single-file. The number of lipids arranged along the translocation coordinate in the pore is not limited in the calculations. The assumption is made that the energetic state of a pore is independent of the sequence of lipids contained in it. The results are valid for an arbitrary number of species with identical kinetic constants. It is shown that infinitely fast diffusion of one vacant site is equivalent to the filled pore approximation, which has been used here. We introduce the concept of non-strict single-file, which allows also for exchanges of neighbouring lipids inside the pore at specified rates. The model successfully simulates the redistribution of lipids between the monolayers of red blood cell plasma membranes under operation of an active aminophospholipid translocase. Kinetic equations are related to linear flux force relations. Phenomenological coefficients are expressed and analysed in terms of kinetic constants. Plausible kinetic pore model parameters are derived from comparison with a reference simulation of a thermodynamic model of the erythrocyte transmembrane lipid distribution. Mechanical forces due to differences in compressions of the lipid molecules between the monolayers are incorporated in kinetic rate constants. It is seen how the active inward transport of aminophospholipids causes an unsymmetrical passive redistribution of the other components due to mechanical effects and cross-coupling of fluxes.

Biological Transport↗

PubMed1996

The kinetics of aflatoxin B1 oxidation by human cDNA-expressed and human liver microsomal cytochromes P450 1A2 and 3A4.

The combined presence of CYP1A2 and 3A4, both of which oxidize aflatoxin B1 (AFB1) to the reactive aflatoxin B1-8,9-epoxide (AFBO) and to hydroxylated inactivation products aflatoxin M1 (AFM1) and aflatoxin Q1 (AFQ1), substantially complicates the kinetic analysis of AFB1 oxidation in human liver microsomes. In the present study, we examine the reaction kinetics of AFB1 oxidation in human liver microsomes (HLMs, N = 3) and in human CYP3A4 and CYP1A2 cDNA-expressed lymphoblastoid microsomes for the purpose of identifying the CYP isoform(s) responsible for AFB1 oxidation at low substrate concentrations approaching those potentially encountered in the diet. AFBO formation by cDNA-expressed human CYP1A2 followed Michaelis-Menten kinetics (Km = 41 microM, Vmax = 2.63 nmol/min/nmol P450). Furthermore, the portion of AFBO formed in HLMs which was eliminated by furafylline, a specific mechanism-based inhibitor of CYP1A2, also followed Michaelis-Menten kinetics (Km = 32-47 microM, Vmax = 0.36-0.69 nmol/min/nmol P450). The formation of AFBO (activation product) and AFQ1 (detoxification product) in cDNA-expressed human CYP3A4 microsomes was sigmoidal and consistent with the kinetics of substrate activation. Accordingly, application of a sigmoid Vmax model equivalent to the Hill equation produced excellent fits to the cDNA-expressed CYP3A4 data and also to the data from HLMs pretreated with furafylline to remove CYP1A2. The Hill model predicted that two substrate binding sites are involved in CYP3A4-mediated AFB1 catalysis and that the average affinity of AFB1 for the two sites was 140-180 microM. Vmax values for AFQ1 formation were 10-fold greater than those for AFBO, and total substrate turnover to both was 67 nmol/min/nmol CYP3A4. Using the derived kinetic parameters for CYP1A2 and 3A4 to model the in vitro rates of AFB activation at low substrate concentrations, it was predicted that CYP1A2 contributes to over 95% of AFB activation in human liver microsomes at 0.1 microM AFB. The important role of CYP1A2 in the in vitro activation of AFB at low substrate concentrations was supported by DNA binding studies. AFB1-DNA binding in control HLMs (reflecting the contribution of CYP1A2 and CYP3A4) and furafylline-pretreated microsomes (reflecting the contribution of CYP3A4 only) catalyzed the binding of 1.71 and 0.085 pmol equivalents of AFB1 to DNA, respectively, indicating that CYP1A2 was responsible for 95% of AFB1-DNA adduct formation at 0.133 microM AFB. These results demonstrate that CYP1A2 dominates the activation of AFB in human liver microsomes in vitro at submicromolar concentrations and support the hypothesis that CYP1A2 is the predominant enzyme responsible for AFBO activation in human liver in vivo at the relatively low dietary concentrations encountered in the human diet, even in high AFB exposure regions of the world. However, because the actual concentrations of AFB in liver in vivo following dietary exposures are uncertain, additional studies in exposed human populations are needed. Quantitative data on the relative rates of AFM1 and AFQ1 excretion (potential biomarkers for CYP1A2 and 3A4 activity, respectively) in humans would be useful to validate the actual contributions of these two enzymes to AFB1 oxidation in vivo.

Adult↗

PubMed2001

Biodegradation kinetics for pesticide exposure assessment.

Understanding pesticide risks requires characterizing pesticide exposure within the environment in a manner that can be broadly generalized across widely varied conditions of use. The coupled processes of sorption and soil degradation are especially important for understanding the potential environmental exposure of pesticides. The data obtained from degradation studies are inherently variable and, when limited in extent, lend uncertainty to exposure characterization and risk assessment. Pesticide decline in soils reflects dynamically coupled processes of sorption and degradation that add complexity to the treatment of soil biodegradation data from a kinetic perspective. Additional complexity arises from study design limitations that may not fully account for the decline in microbial activity of test systems, or that may be inadequate for considerations of all potential dissipation routes for a given pesticide. Accordingly, kinetic treatment of data must accommodate a variety of differing approaches starting with very simple assumptions as to reaction dynamics and extending to more involved treatments if warranted by the available experimental data. Selection of the appropriate kinetic model to describe pesticide degradation should rely on statistical evaluation of the data fit to ensure that the models used are not overparameterized. Recognizing the effects of experimental conditions and methods for kinetic treatment of degradation data is critical for making appropriate comparisons among pesticide biodegradation data sets. Assessment of variability in soil half-life among soils is uncertain because for many pesticides the data on soil degradation rate are limited to one or two soils. Reasonable upper-bound estimates of soil half-life are necessary in risk assessment so that estimated environmental concentrations can be developed from exposure models. Thus, an understanding of the variable and uncertain distribution of soil half-lives in the environment is necessary to estimate bounding values. Statistical evaluation of measures of central tendency for multisoil kinetic studies shows that geometric means better represent the distribution in soil half-lives than do the arithmetic or harmonic means. Estimates of upper-bound soil half-life values based on the upper 90% confidence bound on the geometric mean tend to accurately represent the upper bound when pesticide degradation rate is biologically driven but appear to overestimate the upper bound when there is extensive coupling of biodegradation with sorptive processes. The limited data available comparing distribution in pesticide soil half-lives between multisoil laboratory studies and multilocation field studies suggest that the probability density functions are similar. Thus, upper-bound estimates of pesticide half-life determined from laboratory studies conservatively represent pesticide biodegradation in the field environment for the purposes of exposure and risk assessment. International guidelines and approaches used for interpretations of soil biodegradation reflect many common elements, but differ in how the source and nature of variability in soil kinetic data are considered. Harmonization of approaches for the use of soil biodegradation data will improve the interpretative power of these data for the purposes of exposure and risk assessment.

Biodegradation, Environmental↗

PubMed1982

Kinetic properties of human muscle pyruvate kinase.

The steady-state kinetics of human skeletal muscle pyruvate kinase (MA) and its RNA-complex (MB) has been examined and compared. Kinetic studies revealed significant differences in kinetic properties with respect to free and complex form of pyruvate kinase. The MA form follows a simple Michaelis-Menten kinetics in contrast with the MB form, which displays a negative cooperativity with respect to ADP. Vmax for the complex is 40-60% that for free enzyme. Heterologous RNA is a noncompetitive inhibitor of free enzyme but the kinetics of the complex (MB) is not affected. In presence of 1.0 mM ATP in an assay mixture the kinetic constants of the complex were unchanged except for Vmax, which increased by nearly 60%. Aged preparations of free enzyme (MA) were activated by 100% and more, but the native enzyme was inhibited by 22%. Inorganic phosphate is a potent activator of both forms of pyruvate kinase. In presence of 50 mM K-phosphate the apparent Michaelis constant and interaction coefficient are unchanged, but Vmax for free enzyme increases by 35% and for the complex by 70%, respectively. The specific activity of aged MA form can be restored to the original value after incubation of the enzyme in 50 mM K-phosphate, pH 7.6, or by addition of ATP (1.0 mM) to the assay mixture.

Enzyme Activation↗

PubMed1981

Deconvolution analysis of 99mTc-methylene diphosphonate kinetics in metabolic bone disease.

The kinetics of 99mTc-methylene diphosphonate (MDP) and 47Ca were studied in three patients with osteoporosis, three patients with hyperparathyroidism, and two patients with osteomalacia. The activities of 99mTc-MDP were recorded in the lumbar spine, paravertebral soft tissues, and in venous blood samples for 1 h after injection. The results were submitted to deconvolution analysis to determine regional bone accumulation rates. 47Ca kinetics were analysed by a linear two-compartment model quantitating short-term mineral exchange, exchangeable bone calcium, and calcium accretion. The 99mTc-MDP accumulation rates were small in osteoporosis, greater in hyperparathyroidism, and greatest in osteomalacia. No correlations were obtained between 99mTc-MDP bone accumulation rates and the results of 47Ca kinetics. However, there was a significant relationship between the level of serum alkaline phosphatase and bone accumulation rates (R = 0.71, P less than 0.025). As a result deconvolution analysis of regional 99mTc-MDP kinetics in dynamic bone scans might be useful to quantitate osseous tracer accumulation in metabolic bone disease. The lack of correlation between the results of 99mTc-MDP kinetics and 47Ca kinetics might suggest a preferential binding of 99mTc-MDP to the organic matrix of the bone, as has been suggested by other authors on the basis of experimental and clinical investigations.

Adult↗

PubMed1993

Kinetics of thermal degradation of thiamine and surface colour in canned tuna.

The kinetics of thermal degradation of thiamine and surface colour (lightness measured as Hunter L-value) in canned white tuna were determined using an unsteady-state experimental procedure. Kinetic parameters were calculated by weighted non-linear regression considering a first-order kinetic model with a dependence of the kinetic coefficient (D) with temperature of the Thermal Death Time (TDT) type. Mass-average retentions of thiamine were calculated using a mathematical model which takes into account the non-uniform and unsteady distribution of temperature inside the container during thermal processing. The high correlation obtained between the predicted and the observed retention values and the small confidence intervals found for the kinetic parameters indicate a high statistical reliability. The kinetic model thus determined permits the simulation and optimization of the process resulting in a better quality of the final product.

Animals↗

PubMed1990

Presteady-state kinetics and carrier-mediated transport: a theoretical analysis.

Kinetic studies of cotransport mechanisms have so far been limited to the conventional steady-state approach which does not allow in general to resolve either isomerization or rate-limiting steps and to determine the values of the individual rate constants for the elementary reactions involved along a given transport pathway. Such questions can only be answered using presteady-state or relaxation experiments which, for technical reasons, have not yet been introduced into the field of cotransport kinetics. However, since two recent reports seem compatible with the observation of such transient kinetics, it would appear that theoretical studies are needed to evaluate the validity of such claims and to critically evaluate the expectations from a presteady-state approach. We thus report such a study which was performed on a simple four-state mechanism of carrier-mediated transport. The time-dependent equation for zero-trans substrate uptake was thus derived and then extended to models with p intermediary steps. It is concluded that (p-1) exponential terms will describe the approach to the steady state but that such equations have low analytical value since the parameters of the flux equation cannot be expressed in terms of the individual rate constants of the elementary reactions for models with p greater than 5. We thus propose realistic simplifications based on the time-scale separation hypothesis which allows replacement of the rate constants of the rapid steps by their equilibrium constants, thereby reducing the complexity of the kinetic system. Assuming that only one relaxation can be observed, this treatment generates approximate models for which analytical expressions can easily be derived and simulated through computer modeling. When performed on the four-state mechanism of carrier-mediated transport, the simulations demonstrate the validity of the approximate solutions derived according to this hypothesis. Moreover, our approach clearly shows that presteady-state kinetics, should they become applicable to (co)transport kinetics, could be invaluable in determining more precise transport mechanisms.

Animals↗

PubMedDate not supplied

Cooperative denaturation kinetics of homogeneous polymers.

The kinetics of denaturation of a homogeneous, helical biopolymer with nearest neighbor interactions are described, using a kinetic Ising model in which the configuration of its neighbors dictates the transition probability for a single residue in the chain. The actual kinetics that are simulated using Monte Carlo techniques are compared with the results of analytical kinetic equations for the fraction of helix, (s), generated using the mean-field approximation. This mean-field rate equation is expanded as a hierarchy of terms that characterize the nature of rate constants for interacting systems. The first term in the expansion is first order in (s) and varies linearly with the interaction energy. Subsequent rate terms involve higher powers of (s) and demonstrate the need for nonlinear equations in systems with larger interaction energies. Both the simulations and the mean-field approximation show an intrinsic induction period for the single-step kinetic process. They also yield an apparent first-order rate constant that changes as the reaction proceeds. However, only the simulated kinetics yield ordered regions of chain and a nonzero, nearest-neighbor correlation function.

Kinetics↗

PubMed2000

Assessment of in vivo glucose kinetics using stable isotope tracers to determine their alteration in humans during critical illness.

Glucose plays a central role in energy metabolism, and alterations in its utilization have been reported under a variety of physiological and pathological conditions. The extent and direction of its changes provide useful information to promote the understanding of pathophysiology. The regulation of in vivo glucose kinetics is important because it is closely linked to energy production and the control of amino acid and protein metabolism. Although alterations in glucose kinetics have been demonstrated in critically ill patients, the mechanisms responsible are not well understood. The measurement of glucose kinetics in humans using stable isotopic glucose tracers provides a better understanding of the responses to nutritional support in these patients. While tracer methods have been used to quantitatively measure in vivo kinetics in patients with a variety of critical illnesses as well as in normal volunteers during fasting and exercise in European countries and the United States, they have not received the same attention in Japan. Stable isotopic glucose tracers can be safely given to humans since they are themselves naturally occurring substances, accounting for a small percentage of the total, depending on the isotopic species. The intravenous administration of a glucose tracer allows quantitative assessment of in vivo glucose kinetics under a variety of conditions. This method has wide potential for obtaining kinetic data on all aspects of in vivo glucose metabolism, with major advantages for conducting metabolic studies in humans.

Amino Acids↗

PubMed1999

Bimodal kinetics of a chloride channel from human fibroblasts.

Excised patches were used to study the kinetics of a Cl channel newly identified in cultured human fibroblasts (L132). The conductance of ca. 70 pS in 150 mm symmetrical Cl, and the marked outward rectification ascribe this channel to the ICOR family. Long single-channel recordings (>30 min) revealed that the channel spontaneously switches from a kinetic mode characterized by high voltage dependence (with activity increasing with depolarization; mode 1), into a second mode (mode 2) insensitive to voltage, and characterized by a high activity in the voltage range +/-120 mV. On patch excision the channel always appeared in mode 1, which was maintained for a variable time (5-20 min). In most instances the channels then switched into mode 2, and never were seen to switch back, in spite of the eight patches that cumulatively dwelled in this mode 2.33-fold as compared to mode 1. Stability plots of long recordings showed that the channel was kinetically stable in both modes, allowing standard analysis of steady-state kinetics to be performed. Open and closed time distributions of mode 1 and mode 2 revealed that the apparent number of kinetic states of the channel was the same in the two modes. The transition from mode 1 into mode 2 was not instantaneous, but required a variable time in the range 5-60 sec. During the transition the channel mean open time was intermediate between mode 1 and mode 2. The intermediate duration in the stability plot however is not to be interpreted as if the channel, during the transition, rapidly switches between mode 1 and mode 2, but represents a distinct kinetic feature of the transitional channel.

Cell Line↗

PubMed2006

Fine-tuning of cross-bridge kinetics in cardiac muscle of rat and mouse by myosin light chain isoforms.

Cross-bridge kinetics underlying stretch-induced force transients was studied in cardiac muscle strips with different myosin heavy chain (MHC) and myosin light chain (MLC) isoforms. The force transients were induced by stepwise stretches of maximally Ca(2+)-activated skinned muscle strips. The MHC and MLC isoforms were analyzed by electrophoreses after the mechanical experiments. Muscle strips of euthyroid rats and mice exclusively containing alpha-MHC were used. In addition, muscle strips of hyper- and hypothyroid rats containing different combinations of MHC and MLC isoforms were used. The thyroid hormone is known to alter the expression of MHC but not of MLC isoforms. In muscle strips containing exclusively alpha-MHC, atrial MLC isoforms (all atria of rats and mice) were associated with about 30% faster kinetics than ventricular MLC isoforms (ventricles of hyperthyroid rats and some muscle strips of ventricles of euthyroid rats and mice). On the other hand, in muscle strips containing exclusively ventricular MLC isoforms, alpha-MHC (ventricles of hyperthyroid rats) was associated with about 2.6 times faster kinetics than beta-MHC (ventricles of hypothyroid rats). We conclude that the MLC isoforms fine-tune cross-bridge kinetics, which underlies stretch-induced force transients, whereas the MHC isoforms mainly determine this kinetics. The effect of MLC isoforms on the cross-bridge kinetics may partially contribute to the faster twitch contraction in atria than in ventricles. Furthermore, it may play a role in various cardiomyopathies where atrial MLC isoforms are partially expressed in ventricles or ventricular MLC isoforms are partially expressed in atria.

Animals↗

PubMed2005

Measurement of the kinetics of protein unfolding in viscous systems and implications for protein stability in freeze-drying.

PURPOSE: The aim of the study is to determine the degree of coupling between protein unfolding rate and system viscosity at low temperatures in systems relevant to freeze-drying. METHODS: The cold denaturation of both phosphoglycerate kinase (PGK) and beta-lactoglobulin were chosen as models for the protein unfolding kinetics study. The system viscosity was enhanced by adding stabilizers (such as sucrose), and denaturant (guanidine hydrochloride or urea) was added to balance the stabilizing effect of sucrose to maintain the cold denaturation temperature roughly constant. The protein unfolding kinetics were studied by both temperature-controlled tryptophan emission fluorescence spectroscopy and isothermal high-sensitivity modulated differential scanning calorimetry (MDSC) (Tzero). Viscometers were used to determine the system viscosity. To verify the predictions of structure based on protein unfolding dynamics, protein formulations were freeze-dried above the glass transition temperatures, and the protein structures in dry products were determined by fluorescence spectroscopy of reconstituted solids by extrapolation of the solution data to the time of reconstitution. RESULTS: Empirical equations describing the effect of sucrose and denaturant (urea and guanidine hydrochloride) on protein cold denaturation were developed based on DSC observations [X. C. Tang and M. J. Pikal. The Effects of Stabilizers and Denaturants on the Cold Denaturation Temperature of Proteins and Implications for Freeze-Drying. Pharm. Res. Submitted (2004)]. It was found that protein cold denaturation temperature can be maintained constant in system of increasing sucrose concentration by simultaneous addition of denaturants (urea and guanidine hydrochloride) using the empirical equations as a guide. System viscosities were found to increase dramatically with increasing sucrose concentration and decreasing temperature. The rate constants of protein unfolding (or the half-life of unfolding) below the cold denaturation temperature were determined by fitting the time dependence of either fluorescence spectroscopy peak position shift or DSC heat capacity increase to a first-order reversible kinetic model. The half-life of unfolding did slow considerably as system viscosity increased. The half-life of PGK unfolding, which was only 3.5 min in dilute buffer solution at -10 degrees C, was found to be about 200 min in 37% sucrose at the same temperature. Kinetics of protein unfolding are identical as measured by tryptophan fluorescence emission spectroscopy and by high-sensitivity modulated DSC. The coupling between protein unfolding kinetics and system viscosity for both proteins was significant with a stronger coupling with PGK than with beta-lactoglobulin. The half-lives of PGK and beta-lactoglobulin unfolding are estimated to be 5.5 x 10(11) and 2.2 years, respectively, even when they are freeze-dried in sucrose formulations 20 degrees C above Tg'. Thus, freeze-drying below Tg' should not be necessary to preserve the native conformation. In support of this conclusion, native PGK was obtained after the freeze-drying of PGK at a temperature more than 60 degrees C above the system Tg' in a thermodynamically unstable system during freeze-drying. CONCLUSIONS: Protein unfolding kinetics is highly coupled with system viscosity in high viscosity systems, and the coupling coefficients are protein dependent. Protein unfolding is very slow on the time scale of freeze-drying, even when the system is freeze-dried well above Tg'. Thus, it is not always necessary to freeze-dry protein formulations at temperature below Tg' to avoid protein unfolding. That is, protein formulations could be freeze-dried at product temperature far above the Tg', thereby allowing much shorter freeze-drying cycle times, with dry cake structure being maintained by the simultaneous use of a bulking agent and a disaccharide stabilizer.

Calorimetry, Differential Scanning↗

PubMed1988

Rapid determination of kinetic constants by competitive spectrophotometry.

The use of competitive spectrophotometry to measure kinetic constants for enzyme-catalyzed reactions is described. The equation for the progress curve characterizing the kinetic behavior of an enzyme acting simultaneously on two alternative substrates is derived. By the addition of a competition term to the integrated Michaelis-Menten equation, the kinetic constants of an alternative substrate can be evaluated by measuring the competition with a substrate of known kinetic constants in a single experiment. Studies are presented involving the enzymes leucine aminopeptidase (LAP) and carboxypeptidase A (CPA). The results obtained with LAP and CPA showed that the kinetic constants determined using competitive spectrophotometry were in agreement with values cited in the literature or with values determined by single substrate enzyme kinetics.

Binding, Competitive↗

PubMed1984

Homoserine kinase of Escherichia coli: kinetic mechanism and inhibition by L-aspartate semialdehyde.

The kinetic mechanism of homoserine kinase, purified to homogeneity from Escherichia coli, was examined by initial velocity techniques at pH 7.6. Whereas ATP displayed normal Michaelis-Menten saturation kinetics (Km = 0.2 mM), L-homoserine showed hyperbolic saturation kinetics only up to a concentration of 0.75 mM (Km = 0.15 mM). Above this concentration, L-homoserine caused marked but partial inhibition (Ki approximately 2 mM). The kinetic data indicated that the addition of substrates to homoserine kinase occurs by a preferred order random mechanism, with ATP preferentially binding before L-homoserine. When the ATP concentration was varied at several fixed inhibitory concentrations of L-homoserine, the resulting inhibition pattern indicated hyperbolic mixed inhibition. This suggested a second binding site for L-homoserine. L-Aspartate semialdehyde, an amino acid analog of L-homoserine, proved to be an alternative substrate of homoserine kinase (Km = 0.68 mM), and was subsequently used as a probe of its kinetic mechanism. In aqueous solution, at pH 7.5, this analog was found to exist predominantly (ca 85%) as its hydrated species. When examined as an inhibitor of the physiological reaction, L-aspartate semialdehyde showed mixed inhibition versus both L-homoserine and ATP. Although the pH profiles for the binding of L-homoserine as a substrate (Km) and as an inhibitor (Ki) were identical, the kinetic data were best fit to a two-site model, with separate catalytic and inhibitory sites for L-homoserine.

Adenosine Triphosphate↗

PubMed1978

Flash kinetic study of the last steps in the photoinduced reaction cycle of bacteriorhodopsin.

The reaction cycle of light adapted bacteriorhodopsin (BR) in aqueous purple membrane suspensions was studied by laser flash photolysis at different temperatures (2--49 degrees C) and pH values (3--10). The activation energy for several reaction steps was determined at pH 7.6. The kinetics of O-bacteriorhodopsin (one of the last intermediates in the cycle) were analyzed in some detail and it was found that the simple consecutive reaction scheme M-BR leads to O-BR leads to BR may explain the kinetics of O-bacteriorhodopsin as measured at 680 nm. Since the pH change in neutral aqueous suspensions of purple membrane follows similar kinetics as O-bacteriorhodopsin it is suggested that protons are released during the reaction M-BR leads to O-BR and taken up again during the reacton O-BR leads to BR. Another long-lived intermediate, which absorbs to a greater extent than bacteriorhodopsin at 570 nm and less than bacteriorhodopsin at 420 nm, was identified with the strongly fluorescing species, pseudo- or P-bacteriorhodopsin. The decay of P-bacteriorhodopsin in bacteriohodopsin had an activation energy of only approx. 1.2 kcal/mol, which suggests that the last step of the photocycle is a relaxation around a single bond. At pH 9--10, the simple first-order kinetics of all the intermediates were changed into a kinetics consisting of two first-order decays. This change of kinetics was accompanied by a drastic decrease in the rotational diffusion relaxation time. To explain the results obtained in this work and those of others, a model involving proton uptake and release by the Schiff base nitrogen combined with an isomerization reaction is finally proposed.

Bacteriorhodopsins↗

PubMed1989

A method to distinguish between pore and carrier kinetics applied to urea transport across the erythrocyte membrane.

Permeability coefficients (P) measured at various penetrant concentrations (C) by the perturbation method can be plotted to distinguish simple diffusion, simple pore kinetics and simple carrier kinetics as follows: for simple diffusion, 1/P = constant; for a simple pore, 1/P = 1/Po + 1/Po[1/Kin + 1/Ko]C; for a simple carrier, 1/P = 1/Po + 1/Po[1/Kin + 1/Ko]C + 1/Po[1/(K3K4)] C2 where Po is the maximal permeability at zero penetrant concentration and the K's are combinations of kinetic constants defining each of the transport steps. (Kin and Ko are the half-saturation constant for zero-trans efflux and influx, respectively; K3 is the half-saturation constant for equilibrium exchange, and K4 is related to the mobility of the free carrier). In human erythrocytes, permeability coefficients for diethylene glycol were constant suggesting simple diffusion. For glucose, a plot of 1/P versus concentration was nonlinear indicating carrier kinetics. Plots of 1/P versus penetrant concentrations gave straight lines with positive slopes for urea in human and bovine erythrocytes and for methylurea in human red cells, indicating these penetrants follow simple pore kinetics or simple carrier kinetics in which K4 is very large.

Animals↗