Free energy of subunit interactions. Hemerythrin

Chemical and spectroscopic consequences of allosteric interactions for ligand binding to sipunculid (Phascolopsis gouldii) and brachiopod (Lingula reevii) hemerythrins (Hrs) have been investigated. Possible allosteric effectors for homotropic effects in sipunculid Hrs have been examined, but only reduction in ligand affinity is observed without cooperativity. In contrast to sipunculid Hr, L. reevii Hr binds O2 cooperatively in the pH range 7-8 and exhibits a Bohr effect. Spectroscopic comparisons of the sipunculid and brachiopod Hrs show no significant differences in the active site structures; therefore, modulation of oxygen affinity is attributable to effects linking the site to quaternary structural changes in the octamer. Oxygen equilibria can be fit with a conformational model incorporating a minimum of three states, tensed (T), relaxed (R), and an R-T hybrid. Resonance Raman spectra of L. reevii oxyHr show a shift in the peroxo stretching frequency when the pH is lowered from pH 7.7 (predominantly R oxyHr) to pH 6.3 (a mixture of R, T, and R-T hybrid), but P. gouldii Hr does not have a frequency shift under the same conditions. In contrast to hemoglobins, ligand binding to the deoxy and met forms is noncooperative for brachiopod (and sipunculid) Hrs. It is thus suggested that conformational changes in the protein are linked to the oxidation state change that accompanies oxygenation of the coupled binuclear iron site (deoxy [FeIIFeII]----oxy [FeIIIFeIII]). The total allosteric energy expended in oxygenation is about 1.4 kcal/mol, and such a shift is possible in the relaxed-tense conversion with relatively limited constraints of the iron coordination environment via the protein quaternary structure. The mechanism of cooperativity in the binuclear copper oxygen carrier hemocyanin is discussed in light of these results.

Section: Resultsmentioning

confidence: 99%

Allosteric interactions in sipunculid and brachiopod hemerythrins

Richardson¹,

Emad²,

Reem³

et al. 1987

“…At pH 6.3, 7.5, and 9.0, mixtures of Mes and NaOH, Hepes and NaOH, and Tris and Mes, respectively, were employed. For the anation runs at pH 7.0, where the relative reactivities of monomer and octamer toward SCNwere examined, a buffer of 0.1 M Tris and cacodylic acid was used, in order to simulate the conditions of the molecular weight study (Langerman and Klotz, 1969). Chloride ion in the cacodylic acid was replaced by sulfate ion using an anion exchanger.…”

Section: Methodsmentioning

confidence: 99%

Kinetics of reaction of anions with methemerythrin derivatives

Meloon¹,

Wilkins²

1976

The kinetics of anation of methemerythrin over a wide range of pH and concentration of anions have been studied at 25 degrees C. The azide and thiocyanate ions have been most intensively investigated but experiments with fluoride and chloride are also reported. The replacement of anion in methemerythrin-anionic adducts by other anions has also been studied. Except for replacement of met-fluoride by azide, all replacements can be explained by a dissociative mechanism via the aquated species. Anations are second-order and an associative mechanism is preferred. The second-order rate constant decreases with increasing anion concentrations (from 20 muM to 20 mM). This is attributed to the effect of a secondary anion binding site. The behavior of octameric and monomeric forms of the protein toward thiocyanate is identical. A comparison of results with simple Fe(III) complexes and certain metalloproteins is made.

“…Trimer formation was characterized by an association constant Kx. As in other multimeric protein assemblies, such as the octameric protein hemerythrin (Langerman & Klotz, 1969), definition of an equilibrium constant for the n-mer does not imply that all subunits "simultaneously" interact to form the M-mer but may reflect that equilibrium populations of incomplete assemblies are negligible. Accordingly, K{ is implicitly given by the product KJI^w here K¡¡ is the association constant for dimer formation, is the constant for dimer-trimer equilibrium, and K¿ « K¡¡¡.…”

Section: Determination Ofmentioning

confidence: 99%

Effects of self-association of ornithine aminotransferase on its physicochemical characteristics

Boernke¹,

Stevens²,

Peraino³

1981

Previous work in this laboratory [e.g., Peraino, C., Bunville, L. G., & Tahmisian, T. N. (1969) J. Biol. Chem. 244, 2241--2249, and Morris, J. E., Peraino, C., & Strayer, D. (1974) Proc. Soc. Exp. Biol. Med. 147, 707--709] has shown that the molecular weight of ornithine aminotransferase (OAT) is concentration dependent. In the present study this property of OAT was further characterized by using sedimentation equilibrium centrifugation to determine the molecular weight of OAT in a range of enzyme concentrations. It was shown that OAT aggregates in a two-stage process as its concentration increases. The first stage involves the association of enzymatically active monomers into trimers, with association of the trimers into higher order aggregates occurring in the second stage. Decreasing the pH or raising the ionic strength enhances aggregation while raising the pH inhibits aggregation; however, the two-stage nature of the aggregation process was not affected by changes in pH and ionic strength. Kinetic analyses of purified enzyme showed that aggregation results in an increase in the kM for both substrates with the Vmax remaining constant, indicating that aggregation of monomers sterically hinders substrate binding. Increased Km values were also obtained for OAT sequestered in mitochondria from rats fed a high-protein diet to increase mitochondrial OAT levels. The higher Km values suggest that the elevation of OAT in vivo is accompanied by aggregation of the enzyme within the mitochondrion. We propose that the aggregation-dependent increase of Km in vivo has adaptive value in that it spares ornithine for use in the urea cycle.