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D N Silverman

Publications and source records attributed to D N Silverman.

At least 73 records · Page 4Linked to original sources
PubMed1983

The pH dependence of the hydration of CO2 catalyzed by carbonic anhydrase III from skeletal muscle of the cat. Steady state and equilibrium studies.

We have measured the pH dependence of the kinetics of CO2 hydration catalyzed by carbonic anhydrase III from the skeletal muscle of the cat. Two methods were used: an initial velocity study in which the change in absorbance of a pH indicator was measured in a stopped flow spectrophotometer, and an equilibrium study in which the rate of exchange of 18O between CO2 and H2O was measured with a mass spectrometer. We have found that the steady state constants kCO2 cat and KCO2 m are independent of pH within experimental error in the range of pH 5.0 to 8.5; the rate of release from the enzyme of the oxygen abstracted from substrate HCO-3 in the dehydration is also independent of pH in this range. This behavior is very different from that observed for carbonic anhydrase II for which kCO2 cat and the rate of release of substrate oxygen are very pH-dependent. The rate of interconversion of CO2 and HCO-3 at equilibrium catalyzed by carbonic anhydrase III is not altered when the solvent is changed from H2O to 98% D2O and 2% H2O. Thus, the interconversion probably proceeds without proton transfer in its rate-limiting steps, similar to isozymes I and II.

Animals↗
PubMed1982

Solvent deuterium isotope effects in the catalysis of oxygen-18 exchange by human carbonic anhydrase II.

By measuring the rate of exchange at chemical equilibrium of 18O between HCO3- and H2O catalyzed by human carbonic anhydrase II in the absence of buffers, we have determined the rate of release from the enzyme of water bearing substrate oxygen. The ratio of this rate measured in H2O to the rate measured in D2O, the solvent deuterium isotope effect, is between 4 and 9 in the range of pH(D) from 5.8 to 8.0, with a value of 8.0 +/- 0.7 at pH(D) 6.6 (uncorrected pH meter reading). The magnitude of this isotope effect at pH(D) 6.6 has an exponential dependence on the atom fraction of deuterium in solvent water. We conclude that an intramolecular proton transfer between a proton shuttle group on the enzyme and the active site is rate limiting for the release from the enzyme of water bearing substrate oxygen and involves a change in bonding of more than one proton. In contrast, the solvent deuterium isotope effect on the intermolecular proton transfer between the external buffer imidazole and the active site (or proton shuttle group) of the enzyme is small, 2.3 at pH(D) 7.0, as determined from initial velocity experiments. With a rate constant near 9 X 10(8) M-1 s-1, this intermolecular transfer is limited to a significant extent by diffusion processes.

Carbonic Anhydrases↗
PubMed1982

Carbonic anhydrase activity in mitochondria from rat liver.

An 18O exchange method has been used to determine the location of carbonic anhydrase in mitochondria from rat liver and to examine the role of this enzyme in the kinetics of CO2 in resting and respiring mitochondria. Using digitonin subfractionation, we have determined that a substantial fraction, 40 to 60%, of the carbonic anhydrase activity in the mitochondrion from rat liver is located in the space between the inner and outer membranes; the remaining activity was found in the matrix with no detectable activity in the sedimented membranes. The total catalytic CO2 hydration activity measured in intact mitochondria from rat liver was about 1% of that found in an equal volume of rat erythrocytes. The apparent permeability constant representing the barrier for the diffusion of HCO3(-) from external solution to intramitochondrial carbonic anhydrase, 9 X 10(-5) cm s-1, is near in magnitude to the permeability constant for the diffusion of HCO3(-) across the rat erythrocyte membrane, 4 X 10(-4) cm s-2. Calcium-induced respiratory jumps were shown to cause changes in the rate of 18O exchange between CO2 and H2O that were consistent with a net uptake of CO2 by the mitochondria.

Animals↗
PubMed1981

Diffusion-limited exchange of 18O between CO2 and water in red cell suspensions.

The loss of 18O from labeled CO2 caused by the exchange of oxygen with water, a process catalyzed by carbonic anhydrase, has been measured in suspensions of rat erythrocytes at pH 7.4 and 25 percent C. The rate of loss of 18O from all CO2 and the rate of loss of 18O from doubly-labeled CO2 are shown to be related to the rate constant for the catalyzed hydration of CO2 inside the cell and a rate constant for the diffusion of CO2 out of the cell. The results show that the diffusion of CO2 out of the cell with a half-time near 2 msec is a slower process than the intracellular, catalytic conversion of CO2 to HCO3- which has a half-time near 0.3 msec. From this information we estimate the gradient of 18O content in CO2 in the red cell during an 18O-exchange experiment. The rate constant for the entry of CO2 into red cells, also obtained from 18O-exchange data, has a value of the same magnitude as that anticipated for the diffusion-controlled rate of encounter between CO2 and red cells. This indication of diffusion-controlled depletion of 18O from Co2 is supported by experiments with a carbonic anhydrase inhibitor which show that carbonic anhydrase does not have a rate-limiting role in the 18O exchange until greater than 80% of the enzyme is inhibited.

Animals↗
PubMed1980

Carbon dioxide hydration activity of carbonic anhydrase in mixtures of water and deuterium oxide.

We have measured the hydration of CO2 catalyzed by bovine red cell carbonic anhydrase in mixtures of H2O and D2O at pH(D) which is in the plateau region of the pH--activity profile. The steady-state parameters for hydration Vmax and Km both show an exponential dependence on the atom fraction of deuterium in solvent water. These observed solvent isotope effects cannot be fit to a model involving one hydrogen but must arise from the contributions of two or more hydrogens which change their fractionation factor in the transition state of the rate-limiting step of the catalysis. On the basis of previous evidence, this step is taken to be an intramolecular proton transfer which our results suggest involves water bridges. The dependence of the rate constant for the uncatalyzed reaction between CO2 and water has a nonlinear dependence on the atom fraction of deuterium in the solvent. We conclude that the observed isotope effect must arise from more than one hydrogen in the transition state.

Animals↗
PubMed1979

Role of hemoglobin in proton transfer to the active site of carbonic anhydrase.

The binding of bovine oxyhemoglobin to bovine carbonic anhydrase with a dissociation constant between 10(-5) and 10(-7) M has been determined by countercurrent distribution using aqueous, biphasic polymer systems. This result provides an explanation for the very efficient proton transfer between hemoglobin and carbonic anhydrase, a transfer which enhances the catalytic activity of carbonic anhydrase as measured by 18O exchange between bicarbonate and water at chemical equilibrium (Silverman, D. N., Tu, C. K., and Wynns, G. C. (1978) J. Biol. Chem, 253, 2563-2567). Two rate constants describing 18O exchange activity of carbonic anhydrase at pH 7.5 show saturation behavior when plotted against hemoglobin concentration consistent with a dissociation constant of 2.5 X 10(-6) M between bovine hemoglobin and carbonic anhydrase. Interpretation of these rate constants in terms of a two-step model for 18O exchange indicates that hemoglobin enhances the rate of exchange from carbonic anhydrase of water containing the oxygen abstracted from bicarbonate, but does not affect the catalytic interconversion of CO2 and HCO3- at chemical equilibrium.

Animals↗
PubMed1976

Depletion of 18O from C18O2 in erythrocyte suspensions. The permeability of the erythrocyte membrane to CO2.

The depletion of 18O from CO2, caused by the exchange of oxygen between CO2 and water during the hydration-dehydration cycle, is catalyzed by carbonic anhydrase. This depletion process at chemical equilibrium in the presence of erythrocytes is biphasic, exhibiting a very rapid depletion rate immediately following the addition of cells to an isotonic solution containing 18O-enriched CO2, followed by a much slower depletion rate. It is hypothesized that these depletion characteristics are caused by the diffusion of labeled CO2 into erythrocytes where depletion occurs rapidly due to the large intracellular carbonic anhydrase content. Kinetic equations which describe this hypothesis are solved and a rate constant is obtained which represents the depletion of 18O in CO2 caused by the presence of red cells. These are equilibrium experiments with no net uptake or loss of CO2 in the cells. Consequently, depletion processes are not limited in rate by bicarbonate-chloride exchange or proton distribution across the membrane. The purpose of these measurements is to determine whether the rate of 18O depletion in red cell suspensions is determined by carbonic anhydrase activity in the cell or by the diffusion process by which CO2 enters the cell. This goal is achieved by partially inhibiting carbonic anhydrase with acetazolamide. The rate constant representing 18O depletion caused by the presence of red cells is unchanged, even though up to 90% of carbonic anhydrase is inhibited. From this rate constant the permeability constant of the membrane of rat erythrocytes to CO2 at 25 degrees and pH 7.4 is determined to be (7.6 +/- 1.2) X 10(-3) cm s-1 in the presence of 3.2 mM picrate, a passive anion diffusion inhibitor intended to block HCO3 -flux across the membrane. Using no picrate and allowing HCO3-flux to introduce an error in the measurements, the permeability constant is (1.6 +/- 0.4) X 10(-2) cm s-1. The permeability constants measured by this technique include the diffusion barrier to CO2 not only of the red cell membrane but also of a portion of the intracellular medium.

Animals↗