Publisher Summary The first step toward unravelling the mechanism of an enzyme-catalyzed reaction is to specify the mechanisms available for the reaction concerned. Many of these reactions are not observed when the relevant groups are allowed to come together in bimolecular processes in aqueous solution. For mechanistic work involving intermolecular reactions, it is necessary to use activated substrates. An attractive alternative is to study intramolecular reactions. These are generally faster than the corresponding intermolecular processes. Thus, groups like carboxyl and imidazole are involved at the active sites of many enzymes hydrolyzing aliphatic esters and amides. Therefore, mechanistic studies of intramolecular reactions play an important part in elucidating the chemistry of the groups involved in enzyme catalysis and in defining the mechanisms available for particular reactions.

General acid catalysis and the pH-independent hydrolysis of 2-(p-nitrophenoxy) tetrahydropyran

Effective Molarities for Intramolecular Reactions

Publisher Summary This chapter focuses on vertebrate lysozymes, particularly hen egg-white (HEW) lysozyme. Lysozymes of other types generally are distinguishable from HEW lysozyme in having higher molecular weights and somewhat different enzymic activities. Lysozyme is small and basic, and it separates well on weak acid resins, like Amberlite XE-64 or Bio-Rex 70 or carboxymethyl cellulose and on calcium phosphate gel. Affinity chromatography of lysozyme has been carried out using dispersed chitin or carboxymethyl (CM)-chitin. The conditions required for elution from chitin columns suggest that there are two classes of adsorbent sites that are differently affected by pH and ionic strength in their interaction with the enzyme. The preparation of lysozyme from diverse animal sources is generally achieved in four main steps, which include (1) the preparation of a lysozyme-rich extract, (2) chromatography on CM-cellulose, (3) filtration on Sephadex G-25, and (4) ion exchange chromatography on Amberlite CG-50 at 20° C with a 0.2 M phosphate buffer. The chromatography is sensitive to the pH and small variation of no more than 0.1 pH unit can involve complete retention or exclusion.

General acid catalysis and the pH-independent hydrolysis of 2-(p-nitrophenoxy) tetrahydropyran

Citations

Effective Molarities for Intramolecular Reactions

21 Vertebrate Lysozymes

Efficiency of Proton Transfer Catalysis in Models and Enzymes

Enforced general acid-base catalysis of complex reactions and its limitations

General acid catalysis of acetal, ketal, and ortho ester hydrolysis

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