Time course of exchanges between red cells and extracellular fluid during CO2 uptake.

A stopped-flow rapid-reaction apparatus was used to follow the time course of extracellular pH in a human red cell suspension following a sudden increase in PCO2. The extracellular pH change was slow (t1/2 similar to 3.5 s) considering the presence of carbonic anhydrase in the cells. When carbonic anhydrase was added to the extracellular fluid, the half-time was reduced to less than 20 ms. The explanation for these phenomena is that the equilibration of H+ across the red cell membrane is rate-limited by the uncatalyzed reaction CO2 plus H2O formed from H2CO3 outside the cells. A theoretical model was developed which successfully reproduced the experimental results. When the model was used to simulate CO2 exchange in vivo, it was determined that blood PCO2 and pH require long times (greater than 50 s) to approach equilibrium between cells and plasma after leaving an exchange capillary. We conclude that cell-plasma equilibrium may never be reached in vivo, and that in vitro measurements of these quantities may not represent their true values at the site of sampling.     

Kinetic studies of the choline acetyltransferase reaction using isotope exchange at equilibrium

Isotope exchange at equilibrium has been used to study the kinetic mechanism of the choline acetyltransferase reaction. The choline-acetylcholine, acetyl-CoA-acetylcholine, and CoA-acetyl-CoA exchange patterns are qualitatively consistent with a Theorell-Chance mechanism. However, quantitative differences are observed when the experimental results are compared to theoretical fits of the data for a Theorell-Chance mechanism. It is concluded that the kinetic mechanism of the choline acetyltransferase reaction can best be described as a random Theorell-Chance mechanism in which a low but finite amount of ternary complex exists.     

Observation of two families of folding pathways of BBL

BBL is an independent folding domain of a large multienzyme complex, 2-oxoglutarate dehydrogenase. The folding mechanism of BBL is under debate between the views of noncooperative downhill-type and classical two-state. Extensive replica exchange molecular dynamics simulations of BBL in explicit solvent have shown some non-two-state behaviors despite no definitive evidence of downhill folding. In this work, we postprocess the replica exchange data using our roadmap-based MaxFlux reaction path algorithm to reveal atomically detailed folding pathways. A connected graph is used to organize and visualize the folding pathways initiated from random coils. High structural and transition heterogeneity is seen in the early stage of folding. Two main parallel folding pathways emerge in the later stage; one path shows that tertiary contact and helix formation develop at different stages of folding, whereas the other path exhibits concurrence of secondary and tertiary structure formation to some extent. Because the native state of BBL is sensitive to experimental conditions, we speculate that the relative predominance of the two pathways may vary with the protein construct and solvent conditions, possibly leading to the seeming discrepancy of experimental results. Our roadmap-based reaction path algorithm is a general tool to extract path information from replica exchange.     

The effects of ethidium bromide and Mg++ ion on strand exchange in the Hin-mediated inversion reaction

The biochemical reaction of a site-specific recombinase such as Hin invertase or gammadelta resolvase starts with binding of the recombinase to its recombination site and cleavage of the DNA in the center of the site. This is followed by strand exchange and finally ligation of the ends of the recombined strands. Previous biochemical studies have shown that Hin invertase and gammadelta resolvase cannot proceed beyond DNA cleavage in the absence of Mg++ ion, indicating that these recombinases require Mg++ ion in the strand exchange process. We have observed that the intercalating agent, ethidium bromide (2 microM), does not interfere with DNA cleavage, but slows strand exchange in a concentration-dependent manner. Levels of Mg++ ion below 5 mM also slow strand exchange substantially. We infer that random intercalation of ethidium bromide inhibits unwinding of the double helix at the recombination site in the negatively supercoiled DNA and propose that Mg+ may be required for Hin to deform the secondary structure of B-DNA prior to strand exchange.     

The Exchange of Radioactive Magnesium in the Tissues of the Cow,Calf and Fetus

Calves and pregnant cows were injected with tracer doses of radioactive magnesium and killed at intervals. The pattern of exchange of the isotope with intracellular magnesium in various tissues was broadly similar to that reported for other species except that the equilibration rate was much slower than in the rat. An exception was that the testis magnesium in the 6 week old calves used exchanged more rapidly than in 150 g rats. The exchange of isotope from maternal plasma to fetal tissues was extremely slow.     

Kinetic mechanism of arginyl-tRNA synthetase from human placenta

Like arginyl-tRNA synthetases from other organisms, human placental arginyl-tRNA synthetase catalyzes the arginine-dependent ATP-PPi exchange reaction only in the presence of tRNA. We have investigated the order of substrate addition and product release of this human enzyme in the tRNA aminoacylation reaction by using initial velocity experiments and dead-end product inhibition studies. The kinetic patterns obtained are consistent with a random Ter Ter sequential mechanism, instead of the common Bi Uni Uni Bi ping-pong mechanism for all other human aminoacyl-tRNA synthetases so far investigated in this respect.     

Diffusion and chemical reaction as limiting factors in CO2 equilibration in lungs

Blood CO2 exchange involves at least five separate diffusion and/or chemical reaction processes occurring simultaneously, the rates of several of which have been measured in vitro. Estimation of the influence of the velocity of a single process on the overall rate of CO2 exchange requires calculations using a mathematical model of the system. Computation shows that inasmuch as there is no carbonic anhydrase in plasma, there should be a slow readjustment of plasma pH after blood exchanges CO2 in capillaries. However, there appears to be a carbonic anhydrase in addition to the one in red blood cells that is available to intracapillary fluid in the lung and that accelerates equilibration of the plasma bicarbonate buffer system. This carbonic anhydrase may be in the capillary endothelial cells.     

Mechanisms of enzyme control as probed by equilibrium exchange rates: patterns of modifier effects with a two substrate, two product system

Measurements of reaction rates at equilibrium by isotopic exchange techniques can give considerable information about the mode of action of modifiers of enzymic reaction rates. To illustrate the various patterns that may be obtained, differing effects of modifiers on the exchange of A with P and of B with Q in the simple enzymic reaction of A + BP + Q are given. For this, reasonable values of rate constants are assumed, and calculations made for random and compulsory binding order systems. Cases where modifiers bind at the catalytic sites of substrate or at other binding sites, and where substrate association, substrate dissociation, covalent interconversion, or total catalytic capacity are modified are considered. Some quite distinctive patterns emerge, among the most interesting being those in which a modifier may block net catalysis yet allow one equilibrium exchange to occur essentially unhindered.     

Computer simulation of the P31 NMR equations governing the creatine kinase reaction

A method for solving the Bloch equations that govern magnetization transfer NMR experiments is presented. It requires the numerical evaluation of a matrix exponential and lends itself to computer simulation. It is a simple, versatile method for evaluating P31-NMR magnetization transfer experiments designed to measure biochemical exchange rates. We apply the method to the saturation and inversion transfer experiments and find that the "initial slope" method of determining flux is subject to at least two interpretations. This verifies the accepted concept that biochemical models representing compartmentation and competing reaction hypotheses cannot be reliably distinguished by simply selecting values for NMR and biochemical parameters that give a "best fit" to experimental data. However, our results do indicate that a controlled manipulation of biochemical exchange rate may distinguish between these two models.     

Ligand escape pathways and (un)binding free energy calculations for the hexameric insulin-phenol complex

Cooperative binding of phenolic species to insulin hexamers is known to stabilize pharmaceutical preparations of the hormone. Phenol dissociation is rapid on hexamer dissolution timescales, and phenol unbinding upon dilution is likely the first step in the conversion of (pharmaceutical) hexameric insulin to the active monomeric form upon injection. However, a clear understanding of the determinants of the rates of phenol unbinding remains obscure, chiefly because residues implicated in phenol exchange as determined by NMR are not all associated with likely unbinding routes suggested by the best-resolved hexamer structures. We apply random acceleration molecular dynamics simulation to identify potential escape routes of phenol from hydrophobic cavities in the hexameric insulin-phenol complex. We find three major pathways, which provide new insights into (un)binding mechanisms for phenol. We identify several residues directly participating in escape events that serve to resolve ambiguities from recent NMR experiments. Reaction coordinates for dissociation of phenol are developed based on these exit pathways. Potentials of mean force along the reaction coordinate for each pathway are resolved using multiple independent steered molecular dynamics simulations with second-order cumulant expansion of Jarzynski's equality. Our results for ΔF agree reasonably well within the range of known experimental and previous simulation magnitudes of this quantity. Based on structural analysis and energetic barriers for each pathway, we suggest a plausible preferred mechanism of phenolic exchange that differs from previous mechanisms. Several weakly-bound metastable states are also observed for the first time in the phenol dissociation reaction.     

Subunit interaction during catalysis: ammonium ion modulation of catalytic steps in the Escherichia coli glutamine synthetase reaction

A new approach for assessing if catalytic cooperativity may occur between subunits has been applied to Escherichia coli glutamine synthetase. The extent of oxygen exchange between bound [18O]glutamate and phosphate per molecule of glutamine formed was evaluated at various NH4+ concentrations. This allows calculation of the minimum number of reaction reversals in which bound glutamine is converted to bound glutamate prior to release of glutamine. At 1000 microM NH4+ no detectable reversals occurred, and only one glutamate oxygen appeared in product phosphate as required by the reaction mechanism. However, at 10 microM NH4+ over 15 reversals of bound glutamine formation occurred. Controls showed that under the experimental conditions free glutamine does not become significantly involved in exchange and, therefore, the reversal of the oxygen exchange steps appears to be limited to bound glutamine. In contrast to the effect seen with NH4+, adenosine 5'-triphosphate concentration appears to modulate the exchange of oxygen between glutamate and phosphate only slightly. These findings are interpreted as showing that NH4+ either promotes the dissociation of one of the reaction products or decreases the participation of bound products in the exchange. The NH4+ modulation of the oxygen exchange is consistent with binding of NH4+ at one catalytic site promoting catalytic events at an alternate catalytic site but does not eliminate all other explanations.     

Exchange of recA protein between adjacent recA protein-single-stranded DNA complexes

We have examined the exchange of recA protein between stable complexes formed with single-stranded DNA (ssDNA) and (a) other complexes and (b) a pool of free recA protein. We have also examined the relationship of ATP hydrolysis to these exchange reactions. Exchange was observed between two different recA X ssDNA complexes in the presence of ATP. Complete equilibration between two sets of complexes occurred with a t1/2 of 3-7 min under a set of conditions previously found to be optimal for recA protein-promoted DNA strand exchange. Approximately 200 ATPs were hydrolyzed for every detected migration of a recA monomer from one complex to another. This exchange occurred primarily between adjacent complexes, however. Little or no exchange was observed between recA X ssDNA complexes and the free recA protein pool, even after several hundred molecules of ATP had been hydrolyzed for every recA monomer present. ATP hydrolysis is not coupled to complete dissociation or association of recA protein from or with recA X ssDNA complexes under these conditions.     

Equilibration and exchange of fluorescently labeled molecules in skinned skeletal muscle fibers visualized by confocal microscopy.

Confocal laser fluorescence microscopy was used to study in real time under nearly physiological conditions the equilibration and exchange characteristics of several different fluorescently labeled molecules into chemically skinned, unfixed skeletal muscle fibers of rabbit psoas. The time required for equilibration was found to vary widely from a few minutes up to several days. Specific interactions of molecules with myofibrillar structures seem to slow down equilibration significantly. Time for equilibration, therefore, cannot simply be predicted from diffusion parameters in solution. Specific interactions resulted in characteristic labeling patterns for molecules like creatine kinase (muscle type), pyruvate kinase, actin-binding IgG, and others. For the very slowly equilibrating Rh-NEM-S1, changes in affinity upon binding to actin in the absence of calcium and subsequent slow cooperative activation, beginning at the free end of the filament at the H-zone, were observed. In the presence of calcium, however, binding of Rh-NEM-S1 was homogeneous along the whole actin filament from the very beginning of equilibration. The dissociation properties of the dynamic interactions were analyzed using a chase protocol. Even molecules that bind with rather high affinity and that can be removed only by applying extreme experimental conditions like Rh-phalloidine or Rh-troponin could be displaced easily by unlabeled homologous molecules.     

Evidence for cooperative effects in the exchange reaction catalysed by the oxoglutarate translocator of rat-heart mitochondria.

The initial rates of the exchange external oxoglutarate/internal malate through the inner membrane of rat-heart mitochondria, for various concentrations of the two substrates, have been reinvestigated for an extended range of concentrations of the external oxoglutarate. This has been made possible by use of the inhibitor-stop technique that allows 100 times smaller incubation times than the centrifugation-stop technique used previously. Under the experimental conditions the uptake of the external-labelled oxoglutarate into the mitochondrial-matrix space is mediated by the oxoglutarate translocator performing a ono-to-one exchange of the anions oxoglutarate (external) and malate (internal). Two intermediary-plateau regions are observed in the kinetic saturation curve of the translocator by the external oxoglutarate, revealing a complex rate equation which is found to be the product of two one-substrate functions. Analysing these features it is shown that the model, proposed earlier, of a "double carrier" as catalyst in a rapid-equilibrium random bi-bi mechanism, is still applicable but that several external binding sites have to be considered. As already noticed the external and the internal substrates bind to their respective sites independently of each other. Furthermore, some additional requirements imposed by the observed kinetics suggest that the exchange reaction is performed by only one translocator species made of identical interacting subunits. The anion exchange is tentatively viewed as a rotation of a subunit around an axis situated in the plane of the membrane after two independent local configuration changes induced by the binding of the two substrates on this subunit.     

Effects of perfusate buffer capacity on capillary CO2-HCO3(-)-H+ reactions: theory.

The importance of perfusate nonbicarbonate buffer capacity (beta nonHCO3) to intracapillary CO2-HCO3(-)-H+ reactions was assessed by theoretical analysis of CO2 exchange in saline-perfused pulmonary capillaries. Time courses for perfusate PCO2, [HCO3-], and [H+] were computed for capillaries containing different activities of luminal vascular carbonic anhydrase and different amounts of perfusate nonbicarbonate buffers. Mobilization of perfusate HCO3- toward CO2 during capillary transit is determined by the availability of HCO3- and H+. A supply of protons from the nonbicarbonate buffer pool is necessary to maintain a high rate of HCO3- dehydration. The analyses indicate that beta nonHCO3 has marked nonlinear effects on transcapillary CO2 exchange and intravascular pH equilibration. These nonlinear effects differ from those previously computed for CO2 reactions in an open system because the present model system consists of a sequential combination of open (within capillary proper) and closed (within postcapillary vasculature) systems. The role of luminal vascular carbonic anhydrase in capillary CO2 reactions is strongly dependent on beta nonHCO3. Perfusate nonbicarbonate buffer capacity must be considered when the results of experimental studies of transcapillary CO2 exchange and/or intravascular pH equilibration are interpreted.     

Kinetic analysis of lactose exchange in proteoliposomes reconstituted with purified lac permease

Lactose exchange catalyzed by purified lac permease reconstituted into proteoliposomes was analyzed with unequal concentrations of lactose on either side of the membrane and at low pH so as to prevent equilibration of the two pools. Exchange with external concentrations below 1.0 mM is a single-exponential process, and the apparent affinity constants for external and internal substrate are close to the apparent KMs reported for active transport and efflux, respectively [Viitanen, P.V., Garcia, M. L., & Kaback, H. R. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1629]. At external lactose concentrations above 1.0 mM, a second kinetic pathway becomes evident with an apparent affinity constant of about 6 mM which is similar to the apparent KM for facilitated influx. A second pathway is not observed with respect to internal lactose even when the concentration is increased up to 80 mM. Furthermore, high internal or external lactose concentrations do not inhibit the exchange reaction. Biphasic kinetics with respect to external lactose are retained in a mutant permease that catalyzes exchange but is defective in H(+)-coupled lactose transport. It is suggested that lac permease has more than one binding site and that this may be the underlying reason for the biphasic kinetics observed for both exchange and H(+)-coupled lactose transport.     

Relaxation kinetics of the triple-stranded helix-coil transition at short chain length. A model including the staggering of chains.

The relaxation kinetics of a staggering zipper model are presented, in which the formation of helical nuclei is regarded to be rate-limiting and in which the sliding of strands along the helices is prohibited. Instead a realignment of staggered chains is only possible via complete uncoiling. While maintaining the third order of the initial reaction as in an all-or-none mechanism, the model predicts a large range of relaxation times, which contribute to the mean relaxation time according to the stability of the individual species and which are weakly coupled in the range of small amounts of helical content. The model can easily be compared to experimental results and agrees well with relaxation data obtained from a triple-helical peptide fragment of collagen. It may be readily expanded to other multistranded helix ? coil transitions with steady-state formation of the individual species and it suggests that the fraying of the helix ends is hidden by the fast-relaxation times due to the equilibration of the shortest helical species.     

Electrochemical titration of the cytochrome hemes in the Rhodopseudomonas viridis reaction center. Cyclic equilibrium titrations yield midpoint potentials without evidence for heme cooperativity.

The redox and spectral characteristics of the 4-heme cytochrome c unit of the photochemical reaction center from Rhodopseudomonas viridis were studied by a combination of protein electrochemistry and spectroscopy using an ultra thin-layer spectroelectrochemical cell. Quantitative and reversible reduction of the high-potential and the low-potential hemes was performed in cyclic titrations to record the optical difference spectra in the alpha-band region. The titration of the absorbance from the high-potential hemes can be approximated with a sum of 2 Nernst functions with Em = 0.113 V and Em = 0.175 V. The corresponding titration of the absorbance from the low-potential hemes yielded Em = -0.257 V and Em = -0.175 V (all potentials quoted vs. Ag/AgC1/3 M KCl; add 0.208 V for potentials vs. standard hydrogen electrode). The high-potential hemes equilibrate rapidly and titrate identically for oxidative and reductive titrations. Under identical conditions, the low-potential hemes exhibit a hysteresis, thus indicating much slower equilibration with the applied potential. Cyclic titrations with increasing equilibration periods, however, indicate the disappearance of the hysteresis for equilibration periods approximately twice as long as for the high-potential hemes. We take this as evidence for a slower internal equilibration, but against a cooperativity of the low-potential hemes as observed for other multi-heme cytochromes.