2 – Isolation, Physicochemical Characterization, and Carbohydrate-Binding Specificity of Lectins

Closely related to studying the function of a protein is the analysis of its three-dimensional structure and the identification of interaction sites with its binding partners An alternative approach to the high-resolution methods for three-dimensional protein structure analysis, such as X-ray crystallography and NMR spectroscopy, consists of covalently connecting two functional groups of the protein(s) under investigation The location of the created cross-links imposes a distance constraint on the location of the respective side chains and allows one to draw conclusions on the three-dimensional structure of the protein or a protein complex Recently, chemical cross-linking of proteins has been combined with a mass spectrometric analysis of the created cross-linked products This review article describes the most popular cross-linking reagents for protein structure analysis and gives an overview of the different available strategies that employ chemical cross-linking and different mass spectrometric techniques The challenges for mass spectrometry caused by the enormous complexity of the cross-linking reaction mixtures are emphasized The various approaches described in the literature to facilitate the mass spectrometric detection of cross-linked products as well as computer software for data analyses are reviewed

Chemical cross-linking and mass spectrometry to map three-dimensional protein structures and protein-protein interactions.

Mechanism of interaction of thymidylate synthetase with 5-fluorodeoxyuridylate.

Thymidylate synthetase - a target enzyme in cancer chemotherapy.

Publisher Summary This chapter discusses the results of the studies that permit some generalizations on the catalytic mechanism of glycoside hydrolases from widely differing sources and with different sugar and aglycon specificities and that have become available over the past 15 years. The strong inhibition of glycosidases by aldonolactones was first mentioned in 1940 by Japanese workers who studied β- D -glucosidases from Aspergillus (Taka-diastase) and almonds. Even though both of these groups of compounds are derived from normal substrates by only a minor modification of the glycon moiety, they are discussed together with pseudosubstrates because their reactions with glycosidases show, in many cases, unusual kinetic features. The information relevant to the mechanism of an enzyme-catalyzed reaction can, in general, only be obtained from irreversible inhibitors that react specifically at the active site, and thereby inactivate the enzyme. In many cases, inhibition studies were not carried out to obtain information on the reaction mechanism, but for other purposes. Thus, only inhibitors were tested that were considered suitable for the particular project, for example, studies on the biological function of the enzyme where glycosylamines and aldonolactones are unsuitable.

Glycoside Hydrolases: Mechanistic Information from Studies with Reversible and Irreversible Inhibitors

Evidence for possible nonspecific reactions between N-ethylmaleimide and proteins☆

A unique demonstration is presented of the capacity of glycosidases to create anomeric configuration de novo. Purifed Candida tropicalis alpha-glucosidase and sweet almond beta-glucosidase have been found to attack the same substrate, D-glucal, and to convert this unusual glycosyl substrate (which lacks alpha or beta anomeric configuration) to 2-deoxy-alpha-(or beta-) D-glucose, respectively. The stereospecificity of the hydration reaction catalyzed by each enzyme in D2O was revealed by the use of high-resolution (270 MHz) 1H magnetic resonance spectroscopy. The alpha-glucosidase caused a specific axial protonation (deuteration) of D-glucal at C-2, and formation of 2-deoxy-alpha-D-[2(a)-2H]glucose. The beta-glucosidase catalyzed an oppositely directed axial protonation at C-2 and formation of 2-deoxy-beta-D-[2(e)-2H]glucose. These results are not accounted for by the generally accepted mechanisms of carbohydrase action derived from studies with glycosidically linked substrates alone. D-Glucal apparently binds to the enzymes with essentially the same overall orientation as the D-glucosyl moiety of glycosidically linked substrates (with the double bond of D-glucal lying essentially in the plane of the similarly bound D-glucosyl group). Thus, the alpha-glucosidase evidently protonates D-glucal from above the double bond and alpha-D-glucosidic substrates from below the glycosidic oxygen; beta-glucosidase apparently protonates D-glucal from below the double bond and beta-D-glucosides from above the glycosidic oxygen. A detailed mechanism is proposed for the hydration of D-glucal by each enzyme, involving an incipient glycosyl carbonium ion and assuming the presence at the active site of two carboxyl groups arranged to account for catalysis of glycosylations from glycosidically linked substrates. That D-glucal serves as a glycosyl substrate for these enzymes strongly supports the concept that glycosidases and glycosyltransferases are catalysts of glycosylation (i.e., glycosylases), since this concept does not make the usual assumption that carbohydrases are restricted to acting on substrates having a glycosidic bond and either alph- or beta-anomeric configuration.

Scope and mechanism of carbohydrase action: stereospecific hydration of D-glucal catalyzed by α- and β-glucosidase

Mechanism of binding of mono- and oligosaccharides to concanavalin A: a solvent proton magnetic relaxation dispersion.

Model studies of thymidylate synthetase. Neighboring-group facilitation of electrophilic substitution reactions of uracil furanosides.

A new understanding has been obtained of the catalytic capabilities of trehalase, an enzyme heretofore held to be strictly specific for hydrolyzing alpha, alpha-trehalose and devoid of transglycosylative ability. Highly purified rabbit renal cortical trehalase and a partly purified Candida tropicalis yeast trehalase were found to utilize both alpha- and beta-D-glucosyl fluoride as substrates. In each case, the reactions were competitively inhibited by alpha, alpha-trehalose. Both enzymes catalyzed rapid hydrolysis of alpha-D-glucosyl fluoride to form beta-D-glucose (also, of alpha, alpha-trehalose to form equimolar alpha- and beta-D-glucose). In addition, digests of beta-D-glucosyl fluoride plus alpha-D-[14C]-glucopyranose with either trehalase (but not controls of enzyme with alpha-D-[14C]glucopyranose alone) yielded small amounts of radioactive trehalose (alpha-D-glucopyranosyl alpha-D-[14C]glucopyranoside) which does not accumulate since it is rapidly hydrolyzed. Trehalase thus catalyzes two stereocomplementary types of glycosylation reactions: (I) alpha-D-glucosyl fluoride (or alpha, alpha-trehalose) + H2O leads to beta-D-glucose + HF (or alpha-D-glucose); (II) beta-D-glucosyl fluoride + alpha-D-glucopyranose leads to alpha, alpha-trehalose + HF. Such behavior shows that the catalytic groups of trehalase, as recently found for other glycosylases, are functionally flexible. The results illustrate the inadequacy of conventional views of carbohydrase specificity and the rigor, as a basic guiding principle, of the concept that glycoside hydrolases and glycosyltransferases form a class of glycosylases effecting glycosyl/proton interchange.

Curtis F. Brewer

Papers

Evidence for possible nonspecific reactions between N-ethylmaleimide and proteins☆

Scope and mechanism of carbohydrase action: stereospecific hydration of D-glucal catalyzed by α- and β-glucosidase

Mechanism of binding of mono- and oligosaccharides to concanavalin A: a solvent proton magnetic relaxation dispersion.

Model studies of thymidylate synthetase. Neighboring-group facilitation of electrophilic substitution reactions of uracil furanosides.

Trehalase: stereocomplementary hydrolytic and glucosyl transfer reactions with alpha- and beta-D-glucosyl fluoride.