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J M Yon

Publications and source records attributed to J M Yon.

51 records · Page 3Linked to original sources
PubMed1982

Separation and chemical differentiation of alpha and beta subunits in yeast phosphofructokinase.

The two types of subunits alpha and beta constitutive of yeast phosphofructokinase have been separated by ion-exchange chromatography under denaturating conditions. Amino acid analysis and peptide mapping were performed on the isolated subunits. The frequence of most of the amino acids significantly differs between the two types of polypeptide chains. Moreover, tryptic peptide maps of alpha and beta subunits are clearly not superimposable. These chemical differences seem sufficient to account for the distinct catalytic and regulatory functions of beta and alpha subunits in the yeast phosphofructokinase reaction.

Amino Acids↗
PubMed1979

[Conformational coupling between structural units. A decisive step in the functional structure formation].

Proteins built up of several structural domains are compared with oligomeric proteins made up of several subunits. On this basis the functional consequences of a conformational coupling between domains are analyzed. From the data obtained on the elastase molecule it is proposed that the last step in protein folding insures the optimal coupling between domains and the energy of interaction is utilized for catalysis.

Catalysis↗
PubMed1978

Some aspects of protein folding.

The mechanisms by which a polypeptide chain reaches the three dimensional structure which generates its functional properties are not yet totally understood. The great amount of data now available in the field of protein structure and the data already obtained on protein folding by in vitro experiments allow some schematic representation as, at least, a working hypothesis. In this respect the emphasis is put on the hierarchy in protein structure and the possibility of a folding by stages, some elements or "building blocks" being able to reach independently a native structure and to refine this structure by interacting with each other in the whole molecule.

Concanavalin A↗
PubMed1978

Dependence upon pH of steady-state parameters for the beta-galactosidase-catalysed hydrolyses of beta-D-galactopyranosyl derivatives of different chemical types.

The effect of pH upon the beta-galactosidase-catalyzed hydrolyses of aryl galactosides is essentially similar for each of the three steps of their hydrolysis. It differs markedly from that on the hydrolysis of galactosyl pyridinium salts; these proceed through a 'non-bottleneck' pathway. While pH increase abolishes the rate of every step of the reaction for aryl galactosides, it favors the first step of hydrolysis of the galactosyl pyridinium salts, which supports the hypothesis that catalysis of these compounds originates largely in non-covalent interactions.

Escherichia coli↗
PubMed1978

Conformational adaptability of the active site of beta-galactosidase. Interaction of the enzyme with some substrate analogous effectors.

The action of different effectors, glycosides, and alcohols on the reactions catalyzed by beta-galactosidase is analyzed in this paper. Effectors as large as tri- and tetrasaccharides have no effect on the enzyme activity, suggesting that the binding site has rather small size. Most of the beta-galactosides produce a competitive inhibition. The other compounds assayed behave either as noncompetitive inhibitors, and they are deadened inhibitors, or as uncompetitive inhibitors which exhibit a better affinity for the chemical intermediate than for free enzyme; nearly all of them give transfer products. The analysis of the data indicates that the active center of beta-galactosidase is made up of two subsites: a galactose and a glucose subsite. This latter site is in a more favorable conformation in the galactosylenzyme than in free enzyme; possibly it might even by generated by the galactose binding. Conformational rearrangements of the active center deduced from the inhibition data have been directly observed by differential spectroscopy. The conformational adaptability of the enzyme and its consequence for the functional properties of beta-galactosidase are discussed.

Binding Sites↗
PubMed1978

Functional properties of beta-galactosidase from mutant strain 13 PO of Escherichia coli.

The functional properties of CZP protein, a mutant deriving from wild-type beta-galactosidase (beta-D-galactoside galactohydrolase; EC 3.2.1.23) by a point mutation, were investigated. A large decrease of the specificity, as evaluated by the kcat/Km ratio, was observed, principally originated by a weaker binding of the substrates. The catalytic constants, whose values are strongly affected by the presence of divalent cations, were smaller or larger for mutant enzyme than for wild-type enzyme, depending upon the experimental conditions. Analysis of the kinetic pathway indicates, with some substrates, a change in the limiting step for the mutant enzyme compared to the wild type. Because the k'3 step is rate limiting for hydrolysis of p-nitrophenyl-beta-D-galactoside by the mutant enzyme in the absence of Mg2+ and its value is relatively small, it is possible to observe a burst of p-nitrophenol during hydrolysis. This provides conclusive evidence for the occurrence of a two-step mechanism, with a sequential release of the products.

Cations, Divalent↗
PubMedDate not supplied

Flexibility and folding of phosphoglycerate kinase.

Flexibility and folding of phosphoglycerate kinase, a two-domain monomeric enzyme, have been studied using a wide variety of methods including theoretical approaches. Mutants of yeast phosphoglycerate kinase have been prepared in order to introduce cysteinyl residues as local probes throughout the molecule without perturbating significantly the structural or the functional properties of the enzyme. The apparent reactivity of a unique cysteine in each mutant has been used to study the flexibility of PGK. The regions of larger mobility have been found around residue 183 on segment beta F in the N-domain and residue 376 on helix XII in the C-domain. These regions are also parts of the molecule which unfold first. Ligand binding induces conformational motions in the molecule, especially in the regions located in the cleft. Moreover, the results obtained by introducing a fluorescent probe covalently linked to a cysteine are in agreement with the helix scissor motion of helices 7 and 14 assumed by Blake to direct the hinge bending motion of the domains during the catalytic cycle. The folding process of both horse muscle and yeast phosphoglycerate kinases involves intermediates. These intermediates are more stable in the horse muscle than in the yeast enzyme. In both enzymes, domains behave as structural modules capable of folding and stabilizing independently, but in the horse muscle enzyme the C-domain is more stable and refolds prior to the N-domain, contrary to that which has been observed in the yeast enzyme. A direct demonstration of the independence of domains in yeast phosphoglycerate kinase has been provided following the obtention of separated domains by site-directed mutagenesis. These domains have a native-like structure and refold spontaneously after denaturation by guanidine hydrochloride.

Animals↗
PubMedDate not supplied

The use of inhibitors in the study of glycosidases.

The use of non-covalent as well as covalent inhibitors can be a useful tool to approach the mechanism of activity of glycosidases. An efficient method to determine the essential amino-acid groups directly or indirectly involved in the catalytic process is the use of active site directed irreversible inhibitors. Epoxide derivatives from conduritol B and conduritol C are the most important inhibitors in this group. The use of active site reversible inhibitors: cationic and basic glycosyl derivatives, glycals, glyconolactones, thioglycosides, is effective to study the different charges at the active site or the transition state during catalysis and also to detect conformational adaptability of an enzyme. Furthermore, inhibitors can be valuable tools to investigate various aspects of the physiological role of glycosidases.

1-Deoxynojirimycin↗