No abstract

The mechanism of irreversible thermoinactivation of an enzyme has been quantitatively elucidated in the pH range relevant to enzymatic catalysis. The processes causing irreversible inactivation of hen egg-white lysozyme at 100 degrees C are deamidation of asparagine residues, hydrolysis of peptide bonds at aspartic acid residues., destruction of disulfide bonds, and formation of incorrect (scrambled) structures; their relative contributions depend of the pH.

E-selectin is the inducible adhesion protein on the surface of endothelial cells which has a crucial role in the initial stages of recruitment of leucocytes to sites of inflammation. In addition, it is almost certainly involved in tumor cell adhesion and metastasis. This report is concerned with identification of a new class of oligosaccharide ligand--sulfate-containing--for the human E-selectin molecule from among oligosaccharides on an ovarian cystadenoma glycoprotein. This has been achieved by application of the neoglycolipid technology to oligosaccharides released from the glycoprotein by mild alkaline beta-elimination. Oligosaccharides were conjugated to lipid, resolved by thin-layer chromatography, and tested for binding by Chinese hamster ovary cells which had been transfected to express the full-length E-selectin molecule. Several components with strong E-selectin binding activity were revealed among acidic oligosaccharides. The smallest among these was identified by liquid secondary ion mass spectrometric analysis of the neoglycolipid, in conjunction with methylation analysis of the purified oligosaccharide preparation as an equimolar mixture of the Le(a)- and Le(x)/SSEA-1-type fucotetrasaccharides sulfated at position 3 of outer galactose: [formula: see text] To our knowledge this is the first report of a sulfofucooligosaccharide ligand for E-selectin. The binding activity is substantially greater than those of lipid-linked Le(a) and Le(x)/SSEA-1 sequences and is at least equal to that of the 3'-sialyl-Le(x)/SSEA-1 glycolipid analogue.

The ability to control the resistance of an enzyme to inactivation due to exposure to elevated temperatures is essential for the understanding of thermophilic behavior and for developing rational approaches to enzyme stabilization. By means of site-directed mutagenesis, point mutations have been engineered in the dimeric enzyme yeast triosephosphate isomerase that improve its thermostability. Cumulative replacement of asparagine residues at the subunit interface by residues resistant to heat-induced deterioration and approximating the geometry of asparagine (Asn-14 -* Thr-14 and Asn-78 --Ile-78) nearly doubled the half-life of the enzyme at 100°C, pH 6. Moreover, in an attempt to model the deleterious effects of deamidation, we show that replacement of interfacial Asn-78 by an aspartic acid residue increases the rate constant of irreversible thermal inactivation, drastically decreases the reversible transition temperature, and reduces the stability against dilution-induced dissociation.Enzyme inactivation at elevated temperatures can be divided into two distinct classes (1). Reversible loss of activity is due to disruption of the native conformation above the melting transition; as the name implies, the activity is regained when the enzyme preparation is subsequently cooled. Irreversible loss of activity is the result of deleterious changes in the enzyme structure that are not undone by reducing the temperature.We have recently elucidated the processes causing the irreversible thermoinactivation of enzymes: depending on the pH, inactivating reactions may include deamidation of asparagine residues, hydrolysis of the polypeptide chain at aspartic acid residues, p elimination of cystine residues, as well as certain conformational processes (2). For example, at 100°C and pH 6, hen egg white lysozyme irreversibly inactivates due to a single reaction-deamidation of asparagine residues. The general nature of the mechanisms uncovered in lysozyme has been corroborated in studies on thermostability of bovine pancreatic ribonuclease A (3) and bacterial aamylases (4). Knowledge of the chemical processes responsible for irreversible thermoinactivation affords a straightforward enzyme stabilization strategy: replacement of the "weak links" in the protein molecules with other, more thermoresistant amino acid residues. This strategy was realized in the present study by means of site-directed mutagenesis (5-9). The enzyme used as a model, yeast triosephosphate isomerase (TIM) (Mr 53,000, two identical subunits, no S-S bonds and nonprotein components), has been cloned and expressed in Escherichia coli (10); its complete three-dimensional structure has been determined by x-ray diffraction (11), and site-directed mutagenesis has been successfully implemented for the study of its catalytic properties (12). MATERIALS AND METHODS

We have replaced asparagine residues at the subunit interface of yeast triosephosphate isomerase (TIM) using site-directed mutagenesis in order to elucidate the effects of substitutions on the catalytic activity and conformational stability of the enzyme. The mutant proteins were expressed in a strain of Escherichia coli lacking the bacterial isomerase and purified by ion-exchange and immunoadsorption chromatography. Single replacements of Asn-78 by either Thr or Ile residues had little effect on the enzyme's catalytic efficiency, while the single replacement Asn-78----Asp-78 and the double replacement Asn-14/Asn-78----Thr-14/Ile-78 appreciably lowered kcat for the substrate D-glyceraldehyde 3-phosphate. The isoelectric point of the mutant Asn-78----Asp-78 was equivalent to that of wild-type yeast TIM that had undergone a single, heat-induced deamidation, and this mutant enzyme was less resistant than wild-type TIM to denaturation and inactivation caused by elevated temperature, denaturants, tetrabutylammonium bromide, alkaline pH, and proteases.