Jeannine M. Yon

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.

A preliminary study of the nucleophilic competition in β-galactosidase catalyzed reactions

Abstract Evidence is given for the formation of an intermediary complex following the Michaelis complex in galactoside hydrolysis catalyzed by β-galactosidase. A two step scheme is proposed. The formation of the intermediary complex is the limiting step in phenylgalactoside hydrolysis, while each of the two steps is partially rate controlling in o-nitrophenylgalactoside hydrolysis.

Protein folding in the post‐genomic era

Protein folding is a topic of fundamental interest since it concerns the mechanisms by which the genetic message is translated into the three‐dimensional and functional structure of proteins. In these post‐genomic times, the knowledge of the fundamental principles are required in the exploitation of the information contained in the increasing number of sequenced genomes. Protein folding also has practical applications in the understanding of different pathologies and the development of novel therapeutics to prevent diseases associated with protein misfolding and aggregation. Significant advances have been made ranging from the Anfinsen postulate to the “new view” which describes the folding process in terms of an energy landscape. These new insights arise from both theoretical and experimental studies. The problem of folding in the cellular environment is briefly discussed. The modern view of misfolding and aggregation processes that are involved in several pathologies such as prion and Alzheimer diseases. Several approaches of structure prediction, which is a very active field of research, are described.

The slow-refolding step of phosphoglycerate kinase as monitored by pulse proteolysis

The kinetics of refolding of yeast phosphoglycerate kinase were studied by following the variation in circular dichroism at 218 nm, the recovery of enzyme activity, and the susceptibility to proteolysis by trypsin and V8-protease. A very rapid phase followed by a slower one was detected by circular dichroism, which revealed the formation of secondary structures. The slower phase, with a macroscopic rate constant of 0.35 min-1, was also detected by the susceptibility of the enzyme to both proteases. It was shown that cleavage sites located in the hinge region, in a part of the C-domain and, to a lesser extent, in a region of the N-domain, which are accessible in the intermediate state, became inaccessible during the slow-refolding step of the molecule. These results demonstrate, on the one hand, the role of domains as folding intermediates, and, on the other hand, the locking of the domain structure and the domain pairing that occurs during the slow-refolding step with a rate constant of 0.35 min-1. The return of the enzyme activity occurred in a slower last step upon conformational readjustments induced by domain interactions.

Protein folding in vitro and in the cellular environment

Summary— The main concepts concerning protein folding have been developed from in vitro refolding studies. They state that the folding of a polypeptide chain is a spontaneous process depending only on the amino‐acid sequence in a given environment. It is thermodynamically controlled and driven by the hydrophobic effect. Consequently, it has been accepted that the in vitro refolding process is a valuable model to understand the mechanisms involved during the folding of a nascent polypeptide chain in the cell. Although it does not invalidate the main rules deduced from the in vitro studies, the discovery of molecular chaperones has led to a re‐evaluation of this last point. Indeed, in cells molecular chaperones are able to mediate the folding of polypeptide chains and the assembly of subunits in oligomeric proteins. The possible mechanisms by which these folding helpers act are discussed in the light of the data available in the literature. The folding process is assissted in the cell in different ways, preventing premature folding of the polypeptide chain and suppressing the incorrectly folded species and aggregates. Molecular chaperones bind to incompletely folded proteins in a conformation which suggests that the latter are in the “molten globule” state. However, very little is known about the recognition process.

Regulation of the skeletal muscle metabolism during hibernation of Jaculus orientalis.

1. The glycolytic flow in the skeletal muscle was considerably depressed during hibernation of Jaculus orientalis. 2. Although the ATP content was not modified, the ATP/AMP ratio was twice as large than under homeothermic conditions. 3. Furthermore, the hexose phosphates were markedly depressed. 4. The total activities of the key enzymes, hexokinase, phosphofructokinase and pyruvate kinase, which are regulated through the adenylates, decreased. 5. Under in vivo conditions, taking into account the small amount of fructose-6-phosphate and the ATP/AMP ratio, it is likely that phosphofructokinase was totally inhibited, explaining the undetectable amount of fructose 1.6 bisphosphate.

Survey of the folding pathway of a two-domain protein phosphoglycerate kinase.

The role of structural domains as folding units in the folding process which generates an active enzyme, is considered through several studies on phosphoglycerate kinase, a two-domain enzyme which catalyzes the first step of ATP production in glycolysis. The folding pathway was found to be a complex multi-step process, the C-terminal domain being more stable folding first. Inactive species originating from an intermediate in the folding pathway have been identified. Isolated domains recently obtained using genetic engineering are under investigation in our laboratory; this might probably allow to understand the way by which the N-terminal domain reaches its final native conformation and interacts with the other domain.

Separation and chemical differentiation of α and β subunits in yeast phosphofructokinase

The two types of subunits α and β 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 α and β subunits are clearly not superimposable. These chemical differences seem sufficient to account for the distinct catalytic and regulatory functions of β and α subunits in the yeast phosphofructokinase reaction.

Conformational modification of muscle phosphofructokinase from Jaculus orientalis upon ligand binding

Phosphofructokinase from Jaculus orientalis muscle is an allosteric enzyme regulated by substrates and nucleotide effectors. The conformational modifications upon ligand binding were probed by UV difference spectra and reactivities of thiol groups towards dithiobisnitrobenzoate and N‐ethylmaleimide. The binding of Fru‐6‐P induced significant perturbations in the environment of the aromatic residues and buried the most reactive on the three accessible cysteines per protomer. The same effect on thiol reactivity was observed upon binding of the activator AMP. Various perturbations of both difference spectra and thiol reactivity were detected in the presence of either MG‐ATP, an allosteric inhibitor, or MG‐ITP which is not an effector.

Comparison of muscle phosphofructokinase from euthermic and hibernating Jaculus orientalis. Purification and determination of the quaternary structure

1. The structural properties of skeletal muscle phosphofructokinase from euthermic and hibernating jerboa were compared. 2. The enzyme was purified by a rapid procedure; suspended in ammonium sulfate in the presence of ATP, it was found to be stable for three weeks. 3. A specific activity of 76 U/mg and at most 65 U/mg was obtained for the enzyme from the euthermic and hibernating jerboa, respectively. 4. The molecular weight was estimated to be 320 kDa for the oligomer and 80 kDa for the subunit. 5. A unique alanine residue was found at the C-terminal end, suggesting that the enzyme is a tetramer made of four identical subunits. 6. The tetrameric structure of phosphofructokinase was confirmed by using crosslinking with disuccinimidyl esters. 7. The kinetics of formation of the different crosslinked species were found to be in agreement with a model of the tetramer corresponding to a dihedral symmetry with isologuous contacts between protomers. 8. The same molecular characteristics and immunochemical properties were found for the enzyme extracted from the euthermic and hibernating animals.