It is commonly believed that MgATP2− is the substrate of F1‐ATPases and ATP4− acts as a competitive inhibitor. However, the velocity equation for such competitive inhibition is equivalent to that for ...a rapid equilibrium ordered binding mechanism in which ATP4− adds first and the binding of Mg2+ is dependent on the formation of the E ATP4− complex. According to this ordered‐binding model, solution formed MgATP2− is not recognized by the ATPase as a direct substrate, and the high‐affinity binding of Mg2+ to the E ATP4− complex is the key reaction towards the formation of the ternary complex. These models (and others) were tested with an F1‐ ATPase, isolated from Halobacterium saccharovorum, by evaluating the rate of ATP hydrolysis as a function of free ATP4− or free Mg2+. The rates were asymmetrical with respect to increasing ATP4− versus increasing Mg2+. For the ordered‐binding alternative, a series of apparent dissociation constants were obtained for ATP4− (.cf2.K.cf2..cf1..esapp.rb.eiA.rb), which decreased as Mg2+ increased. From this family of .cf2.K.cf2..cf1..esapp.rb.eiA.rb the true KA was retrieved by extrapolation to Mg2+ = 0 and was found to be 0.2 mM. The dissociation constants for Mg2+, established from these experiments, were also apparent (.cf2.K.cf2..cf1..esapp.rb.eiB.rb) and dependent on ATP4− as well as on the pH. The actual KB was established from a series of .cf2.K.cf2..cf1..esapp.rb.eiB.rb by extrapolating to ATP4− = ∞ and to the absence of competing protons, and was found to be 0.0041 mM. The pKa of the protonable group for Mg2+ binding is 8.2. For the competitive inhibition alternative, rearrangement of the constants and fitting to the velocity equation gave an actual binding constant for MgATP2− (KEAB) of 0.0016 mM and for ATP4− (KEA) of 0.2 mM. Decision between the two models has far‐reaching mechanistic implications. In the competitive inhibition model MgATP2− binds with high affinity, but Mg2+ cannot bind once the E ATP4− complex is formed, while in the ordered‐binding model binding of Mg2+ requires that ATP4− adds first. The steric constraints evident in the diffraction structure of the ATP binding site in the bovine mitochondrial F‐ATPase Abrahams, J. P., Leslie, A. G. W., Lutter, R. & Walker, J. E. (1994) Nature 370, 621−628 tend to favor the ordered‐binding model, but the final decision as to which kinetic model is valid has to be from further structural studies. If the ordered‐binding model gains more experimental support, a revision of the current concepts of unisite catalysis and negative cooperativity of nucleotide binding will be necessary.
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The atomic structure of the light-driven ion pump bacteriorhodopsin and the surrounding lipid matrix was determined by X-ray diffraction of crystals grown in cubic lipid phase. In the extracellular ...region, an extensive three-dimensional hydrogen-bonded network of protein residues and seven water molecules leads from the buried retinal Schiff base and the proton acceptor Asp85 to the membrane surface. Near Lys216 where the retinal binds, transmembrane helix G contains a pi -bulge that causes a non-proline kink. The bulge is stabilized by hydrogen-bonding of the main-chain carbonyl groups of Ala215 and Lys216 with two buried water molecules located between the Schiff base and the proton donor Asp96 in the cytoplasmic region. The results indicate extensive involvement of bound water molecules in both the structure and the function of this seven-helical membrane protein. A bilayer of 18 tightly bound lipid chains forms an annulus around the protein in the crystal. Contacts between the trimers in the membrane plane are mediated almost exclusively by lipids.
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Proline in aqueous solution shows several properties which are unusual for low molecular weight substances. Investigations of solubility, density and viscosity revealed behaviour which is ...characteristic for hydrophilic colloids. 1H-NMR studies indicated a strong hydrogen bonding of water in proline solutions, especially at high concentrations of the solute. From these results it was concluded that proline forms aggregates by stepwise stacking and hydrophobic interaction of the pyrrolidine ring. Thus, the proposed multimer contans a hydrophobic backbone and hydrophilic groups on the surface, exposed to water. Proline solutions are able to increase the solubility of sparingly soluble proteins. The enhancement effect depends on the nature of the protein and on the proline concentration. It is postulated that by a hydrophobic interaction of proline with hydrophobic surface residues of proteins their hydrophilic area is increased. The presence of proline in solutions of the well soluble protein bovine albumin reduces the precipitation of this protein by ethanol and (NH4)2SO4, presumably by an increased water-binding capacity of the proline-protein solution.
The (ν
+ ν
) combination band of water has been investigated in aqueous solutions of proline, glycinebetaine and glycerol and in a three component solution with each of these substances and albumin. ...It is shown that the hydrogen bonding strength between the water protons and proline or betaine is higher than between water and glycerol. Betaine exhibits a higher affinity versus the oxygen of water than does proline.
The water binding capacity of pure proline solutions is unchanged in a three-component solution with albumin. Proline neither enhances nor reduces the solubility of this highly soluble protein. In contrast, in a three-component solution with betaine, the solubility of both betaine and album in is reduced. It is assumed that these solute particles compete for the same binding position on the water molecule. Concentrated glycerol solutions with very low water concentrations dissolve a considerable amount of albumin, which points to the fact that the protein must be partly dissolved in glycerol itself.
In this study, the authors examine the effects of chloride and protons on the chromophores of halorhodopsin and bacteriorhodopsin, with the expectation that these ions, substrates of the two ...light-driven pumps, respectively, will interact with the apoproteins sufficiently to perturb the retinal moiety. All experiments were with cell envelope vesicles at high salt concentrations, which preserved transport activity, i.e., either in 4 M NaCl or in 1.5 M Na sub(2)SO sub(4) with added NaCl. They find that in contrast with the photocycle of bacteriorhodopsin, whose major kinetic component shows sharp pH dependence, the flash-induced absorbance changes in halorhodopsin are virtually independent of pH between 5 and 9. Chloride, on the other hand, which does not affect bacteriorhodopsin under these conditions, has profound effects on the photocycle of halorhodopsin: after addition of chloride, the flash yield for halorhodopsin is greatly enhanced, and the time for recovery from flash bleaching is increased about 4-fold.
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