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H R Bosshard

Publications and source records attributed to H R Bosshard.

At least 73 records · Page 4Linked to original sources
PubMed1982

The cytochrome c peroxidase.cytochrome c electron transfer complex. Experimental support of a hypothetical model.

A hypothetical model of the cytochrome c peroxidase.cytochrome c complex (Poulos, T. L., and Kraut, J. (1980) J. Biol. Chem. 255, 10322-10330) predicts charge interactions between aspartic acid residues of the peroxidase having a spatial distribution that is complementary to the distribution of essential and highly conserved residues of cytochrome c. In a first attempt to test this model, carboxyl groups of cytochrome c peroxidase have been modified with a water-soluble carbodiimide, either alone or in combination with a nucleophile. Modification led to the loss of up to 90% of the ferrocytochrome c peroxidase activity. At least 4-5 carboxyl groups out of a total of 48, but none of the heme carboxyls, were modified in a derivative with 14% residual activity. In the peroxidase.cytochrome c complex the rate of peroxidase inactivation is slowed and approximately 2 carboxyl groups are protected from chemical modification. In the presence of the carbodiimide, cytochrome c and peroxidase were cross-linked to form a covalent 1:1 complex and the linkage sites were preliminarily characterized. Cross-linking occurred to carboxyl groups of the NH2-terminal fragment 1-119 and of fragment 172-229. The four crucial aspartates of the hypothetical model are located in these same two sequence regions.

Animals↗
PubMed1980

The conformation of cytochrome c in solution. Localization of a conformational difference between ferri- and ferrocytochrome c on the surface of the molecule.

The conformation of cytochrome c in solution is believed to change depending on the oxidation-reduction state of the heme iron, since ferri- and ferrocytochrome c exhibit several different physicochemical properties, but so far it is unknown if the conformational difference(s) is (are) confined to a particular part or domain of the molecule. We have therefore applied the method of differential chemical modification (Bosshard, H. R. (1979) Methods Biochem. Anal. 25, 273-304) to compare the chemical reactivity toward acetic anhydride of the 19 lysine residues of ferri- and ferrocytochrome c from horse heart. The epsilon-amino groups of the spatially related residues 39, 53, and 55 were significantly less reactive in ferrocytochrome c as compared to their reactivity in ferricytochrome c. The difference of reactivity was pH-dependent and was shown to be due to an increase of the pK values of the three epsilon-amino groups in ferrocytochrome c. These results indicate a local conformational change on the surface of the cytochrome c molecule in an area to the lower left of and below the heme cleft (standard front view of the molecule facing the exposed edge of the prosthetic group). The local conformational change might be instrumental in the cytochrome c-mediated electron shuttle between cytochrome c1 and a in the final segment of the mitochondrial electron transport chain.

Acetic Anhydrides↗
PubMed1980

Comparison of the binding sites on cytochrome c for cytochrome c oxidase, cytochrome bc1, and cytochrome c1. Differential acetylation of lysyl residues in free and complexed cytochrome c.

The isolated complexes of ferricytochrome c with cytochrome c oxidase, cytochrome c reductase (cytochrome bc1 or complex III), and cytochrome c1 (a subunit of cytochrome c reductase) were investigated by the method of differential chemical modification (Bosshard, H.R. (1979) Methods Biochem. Anal. 25, 273-301). By this method the chemical reactivity of each of the 19 lysyl side chains of horse cytochrome c was compared in free and in complexed cytochrome c and binding sites were deduced from altered chemical reactivities of particular lysyl side chains in complexed cytochrome c. The most important findings follow. 1. The binding sites on cytochrome c for cytochrome c oxidase and cytochrome c reductase, defined in terms of the involvement of particular lysyl residues, are indistinguishable. The two oxidation-reduction partners of cytochrome c interact at the front (exposed heme edge) and top left part of the molecule, shielding mainly lysyl residues 8, 13, 72 + 73, 86, and 87. The chemical reactivity of lysyl residues 22, 39, 53, 55, 60, 99, and 100 is unaffected by complex formation while the remaining lysyl residues in positions 5, 7, 25, 27, 79, and 88 are somewhat less reactive in the complexed molecule. 2. When bound to cytochrome c reductase or to the isolated cytochrome c1 subunit of the reductase the same lysyl side chains of cytochrome c are shielded. This indicates that cytochrome c binds to the c1 subunit of the reductase during the electron transfer process.

Animals↗
PubMed1976

Theories of enzyme specificity and their application to proteases and aminoacyl-transfer RNA synthetases.

The question of enzyme specificity which is a corollary of the phenomenon of biological recognition is reviewed. The following theories are outlined briefly: non-productive binding, induced fit, transition state binding, the general strain theory and the kinetic proofreading hypothesis. Data on proteolytic enzymes and aminoacyl-tRNA synthetases are discussed in the light of predictions made by the various theories. The specificity of inhibitor and substrate binding to chymotrypsin and subtilisins is revealed at the sub-molecular level as an example of binding specificity. Kinetic specificity is experimentally distinguished from binding specificity. Conformational adaptability of enzyme and substrate, which is crucial in some theories, is documented by data on aminoacyl-tRNA synthetases. Expected and observed specificity of tRNA charging is discussed with regard to a theoretical limit of specificity. Additional means seem necessary beside those contained in the isolated enzyme-substrate system to account for the high specificity of most synthetases. In conclusion, we have arrived at quite good explanations for moderate specificity such as is displayed by many proteases, but there are still ample difficulties in the understanding of highly specific enzyme reactions.

Allosteric Regulation↗
PubMed1975

Aminoacyl-tRNA synthetases from Bacillus stearothermophilus. Asymmetry of substrate binding to tyrosyl-tRNA synthetase.

The interaction of L-tyrosine, L-tyrosyladenylate and tRNA-Tyr with tyrosyl-tRNA synthetase from Bacillus stearothermophilus was studied by equilibrium dialysis, gel filtration and fluorescence spectroscopy. The enzyme, which consists of two identical subunits (mol. wt 2 x 44000), binds only a single molecule of L-tyrosine per dimer with a K-d of 2 x 10-5 M at pH 7.8 and 23 degrees C. The tyrosyl-tRNA synthetase--tyrosyladenylate complex which was isolated by gel filtration also has one adenylate bound per dimeric enzyme molecule. In contrast, two tRNA-Tyr molecules bind per enzyme dimer, but the two binding sites are not equivalent having K-d values of 2 x 10-7 M and 1.3 x 10-6 M respectively at pH 6.5 and 25 degrees C. Since crystallographic analysis of the free enzyme [2] shows that the monomer is the asymmetric unit, the data indicate that substrate binding induces asymmetry in the enzyme.

Adenosine Monophosphate↗