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Biomedical subjects

R N Perham

Publications and source records attributed to R N Perham.

At least 199 records · Page 11Linked to original sources
PubMed1976

Selective inactivation of the transacylase components of the 2-oxo acid dehydrogenase multienzyme complexes of Escherichia coli.

1. The reaction of the pyruvate dehydrogenase multienzyme complex of Escherichia coli with maleimides was examined. In the absence of substrates, the complex showed little or no reaction with N-ethylmaleimide. However, in the presence of pyruvate and N-ethylmaleimide, inhibition of the pyruvate dehydrogenase complex was rapid. Modification of the enzyme was restricted to the transacetylase component and the inactivation was proportional to the extent of modification. The lipoamide dehydrogenase activity of the complex was unaffected by the treatment. The simplest explanation is that the lipoyl groups on the transacetylase are reductively acetylated by following the initial stages of the normal catalytic cycle, but are thereby made susceptible to modification. Attempts to characterize the reaction product strongly support this conclusion. 2. Similarly, in the presence of N-ethylmaleimide and NADH, much of the pyruvate dehydrogenase activity was lost within seconds, whereas the lipoamide dehydrogenase activity of the complex disappeared more slowly: the initial site of the reaction with the complex was found to be in the lipoyl transacetylase component. The simplest interpretation of these experiments is that NADH reduces the covalently bound lipoyl groups on the transacetylase by means of the associated lipoamide dehydrogenase component, thereby rendering them susceptible to modification. However, the dependence of the rate and extent of inactivation on NADH concentration was complex and it proved impossible to inhibit the pyruvate dehydrogenase activity completely without unacceptable modification of the other component enzymes. 3. The catalytic reduction of 5,5'-dithiobis-(2-nitrobenzoic acid) by NADH in the presence of the pyruvate dehydrogenase complex was demonstrated. A new mechanism for this reaction is proposed in which NADH causes reduction of the enzyme-bound lipoic acid by means of the associated lipoamide dehydrogenase component and the dihydrolipoamide is then oxidized back to the disulphide form by reaction with 5,5'-dithiobis-(2-nitrobenzoic acid). 4. A maleimide with a relatively bulky N-substituent, N-(4-diemthylamino-3,5-dinitrophenyl)maleimide, was an effective replacement for N-ethylmaleimide in these reactions with the pyruvate dehydrogenase complex. 5. The 2-oxoglutarate dehydrogenase complex of E. coli behaved very similarly to the pyruvate dehydrogenase complex, in accord with the generally accepted mechanisms of the two enzymes. 6. The treatment of the 2-oxo acid dehydrogenase complexes with maleimides in the presence of the appropriate 2-oxo acid substrate provides a simple method for selectively inhibiting the transacylase components and for introducing reporter groups on to the lipoyl groups covalently bound to those components.

Disulfides↗
PubMed1976

Spin-label study of the mobility of enzyme-bound lipoic acid in the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

The lipoic acid residues covalently bound to the transacetylase component of the pyruvate dehydrogenase multienzyme complex of Escherichia coli were selectively modified by reaction with 4-maleimido-2,2,6,6-tetramethylpiperidino-oxyl. The electron-spin-resonance spectrum of the spin-labelled enzyme indicates that the bound nitroxide groups have high mobilities relative to the protein molecule. This physicochemical evidence is consistent with the view that the dithiolane ring of a lipoyl residue is capable of rapid migration between the active sites of the component enzymes in the catalytic mechanism.

Escherichia coli↗
PubMed1975

Self-assembly of biological macromolecules.

The genetic apparatus of the cell is responsible for the accurate biosynthesis of the primary structure of macromolecules which then spontaneously fold up and, in certain circumstances, aggregate to yield the complex tertiary and quaternary structures of the biologically active molecules. Structures capable of self-assembly in this range from simple monomers through oligomers to complex multimeric structures that may contain more than one type of polypeptide chain and components other than protein. It is becoming clear that even with the simpler monomeric enzymes there is becoming clear that even with the simpler monomeric enzymes there is a kinetically determined pathway for the folding process and that a folded protein must now be regarded as the minimum free energy form of the kinetically accessible conformations. It is argued that the denatured subunits of oligomeric enzymes are likely to fold to something like their final structure before aggregating to give the native quaternary structure and the available evidence would suggest that this is so. The importance of nucleation events and stable intermediates in the self-assembly of more complex structures is clear. Many self-assembling structures contain only identical subunits and symmetry arguments are very successful in accounting for the structures formed. Because proteins are themselves complex molecules and not inelastic geometric objects, the rules of strict symmetry can be bent and quasi-equivalent bonding between subunits permitted. This possibility is frequently employed in biological structures. Conversely, symmetry arguments can offer a reliable means of choosing between alternative models for a given structure. It can be seen that proteins gain stability by growing larger and it is argued in evolutionary terms that aggregation of subunits is the preferred way to increase the size of proteins. The possession of quaternary structure by enzymes allows conferral of other biologically important properties, such as cooperativity between active sites, changes of specificity, substrate channelling and sequential reactions within a multi-enzyme complex. Comparison is made of the invariant subunit compositions of the simpler oligomeric enzymes with the variation evidently open to, say, the 2-oxoacid dehydrogenase complexes of E. coli. With viruses, on the other hand, the function of the quaternary structure is to package nucleic acid and, as an example, the assembly and breakdown of tobacco mosaic virus is discussed. Attention is drawn to the possible ways in which the principles of self-assembly can be extended to make structures more complicated than those that can be formed by simple aggregation of the comonent parts.

Binding Sites↗
PubMed1974

An amino acid sequence in the active site of lipoamide dehydrogenase from pig heart.

1. The two cysteine residues forming the disulphide bridge that comprises part of the active site of lipoamide dehydrogenase from pig heart were specifically labelled with iodo[2-(14)C]acetic acid. 2. A tryptic peptide containing these carboxymethylcysteine residues was isolated from digests of reduced and S-carboxymethylated lipoamide dehydrogenase and its amino acid sequence of 23 residues was determined. 3. The sequence is highly homologous with a similar sequence containing the active-site disulphide bridge of lipoamide dehydrogenase derived from the 2-oxoglutarate dehydrogenase complex of Escherichia coli (Crookes strain) and it is probable that, as in the bacterial enzyme, the disulphide bridge forms an intrachain loop containing six residues. The results indicate that the bacterial and mammalian proteins have a common genetic origin. 4. Amino acid sequences containing six other unique carboxymethylcysteine residues were also partly determined. 5. The analysis of the primary structure thus far is consistent with the view that the enzyme (mol.wt. approx. 110000) is composed of two identical polypeptide chains.

Amino Acid Sequence↗
PubMed1974

Kinetic and molecular properties of citraconyl-aldolase. The reversible denaturation and hybridization of the native and modified enzymes.

1. The preparation of enzymically active N-citraconyl derivatives of fructose diphosphate aldolase from rabbit muscle is described. Reaction is restricted to amino groups and the derivatives are not very heterogeneous with respect to the number of substituents. 2. Linear double-reciprocal plots of enzyme velocity against substrate concentration are found up to about 15% blocking of amino groups. With more than 15% blocking, there is a marked downward curvature in the double-reciprocal plots at high substrate concentrations. 3. Over the range 0-25% blocking of amino groups the apparent V(max.) for fructose diphosphate falls to 10% that of the native enzyme, and the apparent K(m) rises from 1 to 400mum. 4. Various pieces of evidence suggest that citraconyl-aldolase is slightly distorted in structure compared with the native enzyme. However, the kinetic properties and tetrameric structure of citraconyl-aldolase can be completely recovered after denaturation in 4m-guanidine hydrochloride. 5. After removal of the citraconyl groups in acid conditions the kinetic and molecular properties of native enzyme are restored. 6. Hybrid forms of aldolase can be constructed containing native and citraconylated subunits and the suitability of these derivatives for the study of subunit interactions in the enzyme is discussed. 7. The kinetic properties of hybridized aldolase containing native and citraconylated subunits are not exactly those predicted from the kinetic properties of the two parental forms. This result is interpreted in terms of conformational changes induced in the native and modified subunits when both are present in a hybrid molecule, evidently as a result of interactions in the tetramer.

Acylation↗
PubMed1973

The reactivity of functional groups as a probe for investigating the topography of tobacco mosaic virus. The use of mutants with additional lysine residues in the coat protein.

Several mutants of tobacco mosaic virus that contain additional lysine residues as a result of mutations in the coat protein were investigated. Mutant E66 has a lysine residue replacing asparagine at position 140 when compared with the wild-type vulgare and this lysine residue reacts readily in the intact virus with methyl picolinimidate. Mutant B13a has two new lysine residues in the coat protein, replacing a glutamine at position 9 and an asparagine at position 33, whereas mutant B13b has the single replacement of glutamine by lysine at position 9. The lysine residue at position 9 in mutants B13a and B13b also reacts readily with methyl picolinimidate in the intact virus but the lysine at position 33 in mutant B13a did not react under these conditions. However, when the isolated coat protein from mutant B13a was treated with methyl picolinimidate, the lysine residue at position 33 did become modified, showing that the loss in reactivity of this residue towards the imidoester in the intact virus is a result of the assembly of the protein subunit into the virus structure. These results are compatible with and extend previous studies on the sero-logical properties of mutants of tobacco mosaic virus and illustrate the value of methyl picolinimidate as a reagent for probing the accessibility of amino groups in proteins. When intact tobacco mosaic virus (vulgare) was treated with p-iodobenzenesulphonyl chloride, no reaction with the lysine residues at positions 33 or 68 in the virus subunit could be detected but complete modification of tyrosine-139 was achieved. This result also extends previous studies with other reagents. The usefulness of the differential reactivity of the lysine residues in tobacco mosaic virus and its mutants as a means of attaching heavy-atom labels at chemically defined positions for subsequent X-ray-diffraction analysis and the implications of these experiments for deciphering the folding of the peptide chain in the virus subunit are discussed.

Amino Acid Sequence↗
PubMed1973

The reaction of protein amino groups with methyl 5-iodopyridine-2-carboximidate. A possible general method of preparing isomorphous heavy-atom derivatives of proteins.

1. The synthesis of methyl 5-iodopyridine-2-carboximidate and its reaction with amino groups of model compounds and performic acid-oxidized insulin are described. The reagent was designed to introduce heavy atoms into specific sites in proteins. 2. Specific reaction with the amino groups of oxidized insulin can be achieved under reasonably mild conditions giving rise to the corresponding N-monosubstituted amidines. 3. The extent of reaction of this reagent with protein amino groups can be readily determined by difference spectroscopy. Modification of lysine residues inhibits tryptic cleavage at such residues, and this can be of assistance in establishing the site of modification in the primary structure. 4. Evidence is presented to show that methyl 5-iodopyridine-2-carboximidate can react specifically, at pH5.0, with the aromatic amino group of 3-amino-l-tyrosine; the final product of this reaction is a 2-arylbenzoxazole. 5. The use of this reagent as a general method for preparing heavy-atom isomorphous derivatives of proteins is discussed.

Amidines↗
PubMed1973

Purification and characterization of two fructose diphosphate aldolases from Escherichia coli (Crookes' strain).

Two fructose diphosphate aldolases (EC 4.1.2.13) were detected in extracts of Escherichia coli (Crookes' strain) grown on pyruvate or lactate. The two enzymes can be resolved by chromatography on DEAE-cellulose at pH7.5, or by gel filtration on Sephadex G-200, and both have been obtained in a pure state. One is a typical bacterial aldolase (class II) in that it is strongly inhibited by metal-chelating agents and is reactivated by bivalent metal ions, e.g. Ca(2+), Zn(2+). It is a dimer with a molecular weight of approx. 70000, and the K(m) value for fructose diphosphate is about 0.85mm. The other aldolase is not dependent on metal ions for its activity, but is inhibited by reduction with NaBH(4) in the presence of substrate. The K(m) value for fructose diphosphate is about 20mum (although the Lineweaver-Burk plot is not linear) and the enzyme is probably a tetramer with molecular weight approx. 140000. It has been crystallized. On the basis of these properties it is tentatively assigned to class I. The appearance of a class I aldolase in bacteria was unexpected, and its synthesis in E. coli is apparently favoured by conditions of gluconeogenesis. Only aldolase of class II was found in E. coli that had been grown on glucose. The significance of these results for the evolution of fructose diphosphate aldolases is briefly discussed.

Amino Acids↗