Igor W. Plesner

Enantiospecific synthesis of 1-azafagomine.

For the first time the two enantiomeric forms of the glycosidase inhibitor 1-azafagomine have been synthesised starting from D- and L-xylose. D-Xylose was converted to the 2,3,5-tribenzylfuranose, which upon reductive amination with tert-butyl carbazate gave the protected 1-hydrazino-1-deoxypentitol in high yield. N-acetylation, mesylation of the 4-OH, removal of the Boc group, cyclisation and deprotection gave (+)-1-azafagomine ((+)-1). By a similar sequence of reactions, L-xylose was converted to (-)-1-azafagomine ((-)-1). Enzymatic and other routes to optically pure 1-azafagomine were also studied. Compound (-)-1 is a potent competitive glycosidase inhibitor, while (+)-1 has no biological activity. The inhibition of almond beta-glucosidase by (-)-1 was found to be slow owing to a slow binding step of inhibitor to enzyme, with no subsequent conformational rearrangement. The rate constants for binding and release were found to be 3.3 x 10(4)M(-1)s(-1) and 0.011 s(-1), respectively, yielding Ki = 0.33 microM.

The steady-state kinetic mechanism of ATP hydrolysis catalyzed by membrane-bound (Na+ + K+)-ATPase from ox brain IV. Rate constant determination

Abstract The expressions for the kinetic constants corresponding to the steady state model for hydrolysis of ATP catalyzed by (Na + + K + )-ATPase proposed recently are analyzed with the object of determining the rate constants. The theoretical background for the necessary procedures is described. The results of this analysis are: (1) A small class (four) of rate constants are determined directly by the previously published values of the kinetic constants. (2) For a somewhat larger class of rate constants upper and lower bounds may be established. For several rate constants the upper and lower bounds differ by less than a factor 1.6 (for the ‘(Na + + K + )-enzyme’, i.e. the enzyme activity with K + and millimolar substrate concentration) and 1.2 (for the ‘Na + -enzyme’, i.e. the activity at micromolar substrate concentrations). (3) Experiments on inhibition by K + of the Na + -enzyme at various Mg 2+ concentrations are reported and analyzed. With the additional assumption that the rate constants governing the addition to ATP of Mg 2+ is independent of whether or not ATP is bound to an enzyme molecule, a set of consistent values for all the 23 rate constants in the mechanism may be obtained. (4) The values of some rate constants lend further support to the contention discussed in a previous paper that the enzyme hydrolyzes ATP along two kinetically distinct pathways, depending on the presence of K + and on the concentration of substrate, without the necessity of having more than one active substrate site per enzyme unit at any time. (5) The results show that while the two enzyme forms, the ‘Na + -enzyme’ E 1 and the “K + -enzyme” E 2 K, add substrate with (second order) rate constants of the same order of magnitude (differing only by a factor of four in favor of the former), the rate constants for the reverse processes differ by a factor of 100, being largest for the K + -enzyme. This is the main reason for the large difference in the Michaelis constants for the two forms reported previously. (6) Compatibility of the model with the well-known rapid dephosphorylation of the phosphorylated enzyme in the presence of K + requires the presence, at non-zero steady state concentration, of an enzyme-potassium-phosphate intermediate, which is acid labile and is therefore not detected as a phosphorylated enzyme using conventional methods.

Kinetics of (Na++K+)-ATPase: analysis of the influence of Na+ and K+ by steady-state kinetics

The influence of Na+ and K+ on the steady-state kinetics at 37 degrees C of (Na+ + K+)-ATPase was investigated. From an analysis of the dependence of slopes and intercepts (from double-reciprocal plots or from Hanes plots) of the primary data on Na+ and K+ concentrations a detailed model for the interaction of the cations with the individual steps in the mechanism may be inferred and a set of intrinsic (i.e. cation independent) rate constants and cation dissociation constants are obtained. A comparison of the rate constants with those obtained from an analogous analysis of Na+-ATPase kinetics (preceding paper) provides evidence that the ATP hydrolysis proceeds through a series of intermediates, all of which are kinetically different from those responsible for the Na+-ATPase activity. The complete model for the enzyme thus involves two distinct, but doubly connected, hydrolysis cycles. The model derived for (Na+ + K+)-ATPase has the following properties: The empty, substrate free, enzyme form is the K+-bound form E2K. Na+ (Kd = 9 mM) and MgATP (Kd = 0.48 mM), in that order, must be bound to it in order to effect K+ release. Thus Na+ and K+ are simultaneously present on the enzyme in part of the reaction cycle. Each enzyme unit has three equivalent and independent Na+ sites. K+ binding to high-affinity sites (Kd = 1.4 mM) on the presumed phosphorylated intermediate is preceded by release of Na+ from low-affinity sites (Kd = 430 mM). The stoichiometry is variable, and may be Na:K:ATP = 3:2:1. To the extent that the transport properties of the enzyme are reflected in the kinetic ATPase model, these properties are in accord with one of the models shown by Sachs ((1980) J. Physiol. 302, 219-240) to give a quantitative fit of transport data for red blood cells.

Slow inhibition of almond β-glucosidase by azasugars: determination of activation energies for slow binding

The thermodynamic and activation energies of the slow inhibition of almond beta-glucosidase with a series of azasugars were determined. The inhibitors studied were isofagomine ((3R,4R,5R)-3,4-dihydroxy-5-hydroxymethylpiperidine, 1), isogalactofagomine ((3R,4S,5R)-3,4-dihydroxy-5-hydroxymethylpiperidine, 2), (-)-1-azafagomine ((3R,4R,5R)-4,5-dihydroxy-3-hydroxymethylhexahydropyridazine, 3), 3-amino-3-deoxy-1-azafagomine (4) and 1-deoxynojirimycin (5). It was found that the binding of 1 to the enzyme has an activation enthalpy of 56.1 kJ/mol and an activation entropy of 25.8 J/molK. The dissociation of the enzyme-1 complex had an activation enthalpy of -2.5 kJ/mol and an activation entropy of -297 J/molK. It is suggested that the activation enthalpy of association is due to the breaking of bonds to water, while the large negative activation entropy of dissociation is due at least in part to the resolvation of the enzyme with water molecules. For the association of 1 DeltaH(0) is 58.6 kJ/mol and DeltaS(0) is 323.8 J/molK. Inhibitor 3 has an activation enthalpy of 39.3 kJ/mol and an activation entropy of -17.9 J/molK for binding to the enzyme, and an activation enthalpy of 40.8 kJ/mol and an activation entropy of -141.0 J/molK for dissociation of the enzyme-inhibitor complex. For the association of 3 DeltaH(0) is -1.5 kJ/mol and DeltaS(0) is 123.1 J/molK. Inhibitor 5 is not a slow inhibitor, but its DeltaH(0) and DeltaS(0) of association are -30 kJ/mol and -13.1 J/molK. The large difference in DeltaS(0) of association of the different inhibitors suggests that the anomeric nitrogen atom of inhibitors 1-4 is involved in an interaction that results in a large entropy increase.

Direct NMR-Spectroscopic Determination of Active-Enzyme Concentration by Titration with a Labeled Inhibitor: Determination of thekcat Value of Almondβ-Glucosidase

A new method for the determination of active‐enzyme concentration of a glucosidase by using 13C NMR spectroscopy is reported. The method consists of quantifying the binding between a 13C‐labelled, strong competitive inhibitor, [5‐13C]‐1‐azafagomine (1), and the enzyme. The concentration of free inhibitor 1 is measured in a series of binding experiments from the intensity of its NMR signal relative to that of a reference. From a plot of the concentrations of bound vs. free inhibitor 1, the amount of specifically bound 1, that is, the amount of active sites, is determined. From this value, active‐enzyme concentration and kcat value can be calculated.

Phase separation in monolayers of adsorbed ions

Numerical calculations are performed on a dilute electrolyte solution in equilibrium with a charged surface, using the theory developed by Buff and Stillinger. The concentrations of adsorbed cations in the double layer (in the case of a negatively charged surface) are computed as a function of the electrostatic potential in the adsorption zone. It is found that a critical value of the potential exists, such that, for values of the potential more negative than the critical value, a phase transition in the double layer occurs to yield an almost close‐packed layer of counterions. The results are discussed in the light of similar effects found in polyelectrolyte theory as well as of experimental results from nuclear relaxation studies. It is demonstrated that in the case of a mixed electrolyte solution (two monovalent cations and a common monovalent anion) the adsorbing surface will selectively adsorb the smallest cation below the threshold potential, even though the larger ion may predominate in the bulk pha...

A model of mitochondrial creatine kinase binding to membranes: adsorption constants, essential amino acids and the effect of ionic strength

The quantitative aspects of mitochondrial creatine kinase (mitCK) binding to mitochondrial membranes were investigated. A simple adsorption and binding model was used for data fitting, taking into account the influence of protein concentration, pH, ionic strength and substrate concentration on the enzyme adsorption. An analysis of our own data as well as of the data from the literature is consistent with the adsorption site of the octameric mitCK being composed of 4 amino acid residues with pK = 8.8 in the free enzyme. The pK value changes to 9.8 upon binding of the protein to the membrane. Lysine is suggested as the main candidate to form the adsorption site of mitCK. Deprotonated octameric mitCK easily dissociated from the membrane (Ka = 0.39 mM at ionic strength I = 7.5 mM and 5 degrees C); after protonation its affinity increased many times (Kah = 39 nM). Determination of mitCK adsorption capacity by another method at pH 7.4, when the enzyme is almost protonated, gave Kah = 15 nM. The effect of ionic strength on mitCK adsorption may be described in terms of Debye-Hückels theory for activity coefficients assuming the charges of the interacting species to be +4 and -4. The dissociation constant for the mitCK-membrane complex at pH 7.4 and I = 0 was evaluated by different approaches as approx. 1 nM. Extramitochondrial ATP (or ADP) shifted greatly the equilibrium between the adsorbed and the free mitCK towards the solubilized state, since in the adsorbed protein the external ligands had access to four binding sites and in the free protein to eight sites.

Kinetics of oligomycin inhibition and activation of Na+/K(+)-ATPase.

Oligomycin inhibition of the maximal hydrolysis activity of ox brain Na+/K(+)-ATPase was studied at varying NaCl concentrations and it was found that for a given amount of live enzyme, the observed inhibition of a particular total oligomycin concentration decreased as the amount of added, (heat-) denatured enzyme increased. In the present article we derive a scale factor for the oligomycin concentration, i.e., the fraction of the total concentration of oligomycin which is free in solution, as a function of the enzyme concentration used. This fraction decreased linearly with the protein concentration and may attain quite small values. We also study the Na(+)-dependence of the hydrolysis rate at saturating substrate concentrations ([Mg2+] = [ATP] = 3 mM), in the presence as well as the absence of KCl, at various concentrations of oligomycin. These data may be explained if it is assumed that the sole effect of oligomycin is to confer upon the enzyme an increased affinity for Na+, i.e., oligomycin merely enhances the inhibitory effect of Na+ on the (maximal) activity seen at high Na(+)-concentrations. The increased Na(+)-affinity in the presence of oligomycin should result in activation of the hydrolysis rate measured under conditions where Na(+)-activation is predominant, i.e., at low Na(+)-concentration and sub-saturating substrate concentrations. This prediction is verified for both Na(+)-ATPase and for Na+/K(+)-ATPase. This proposed action of oligomycin seems to be corroborated also by other evidence discussed in the text.

Distinction between the intermediates in Na+-ATPase and Na+, K+-ATPase reactions. I: Exchange and hydrolysis kinetics at millimolar nucleotide concentrations

Parallel measurements in steady-state of ATP hydrolysis rate (vhydr) and the simultaneous reverse reaction, i.e., the ADP-ATP exchange rate (vexch), allowed the determination of a kinetic parameter, KE, containing only the four rate constants needed to characterize the enzyme intermediates involved in the sequence (Formula: see text). In order to compare the properties of these enzyme intermediates under different sets of conditions, KE was measured at varying K+ and Na+ concentrations in the presence of millimolar concentrations of ATP, ADP and MgATP, using an enzyme preparation that was partially purified from bovine brain. (1) In the presence of Na+ (150 mM), K+ (20-150 mM) was found to increase the exchange rate and decrease the ATP hydrolysis rate at steady-state. As a result, KE increased at increasing K+. However, the value of KE found by extrapolation to K+ = 0 was 7-times lower than the value actually measured in the absence of K+. This finding indicates that one of the intermediates, EATP or EP, or both, when formed in the presence of Na+ alone, are different from the corresponding intermediate(s) formed in the presence of Na+ + K+ (at millimolar substrate concentration). (2) In the presence of 150 mM K+, Na+ (5-30 mM) was found to increase the ADP/ATP exchange as well as the ATP hydrolysis rate at steady-state. The ratio of the two rates was constant. This finding, when interpreted in terms of KE, indicates that Na+ does not have to leave the enzyme for ATP release to be accelerated by K+ in the backward reaction. This also is in opposition to the usual versions of the Albers-Post model, which does not have simultaneous presence of Na+ and K+.

Two unexplained kinetic features of NA,K-ATPase may be understood as indicating K(+)-induced cooperativity between subunits in a dimeric enzyme.

In 1994 Klodos et al.’ published a detailed study of the transient dephosphorylation kinetics of Na,K-ATPase as a function of the NaCl concentration in the dephosphorylating medium (chase solution). Experiments were performed both by initiating the dephosphorylation by the addition of ADP and by adding K+. Most experiments were performed at 0°C. On the basis of their experiments the authors concluded that new features in a model, such as “heterogeneous kinetics,” that is, processes involving several unmixed lipid phases, or, alternatively, sudden temporary changes in rate constants on changing the salt concentration and subsequent relaxation to the initial values, were necessary to interpret the results. In 1995 Schwarzbaum et aL2 observed that although the steady-state velocity of the enzyme divided by the total phosphoenzyme concentration in the absence of K + was independent of the ATP concentration, a property characteristic of the Post-Albers model, that quantity was a hyperbolic function of ATP in the presence of K+. These two sets of properties are interpreted in this presentation.