Desensitization of allosteric sites: consequences on the conformation and catalytic properties of regulatory enzymes

Abstract

Regulatory enzymes are characterized by their interactions with molecules which are structurally unrelated to their substrates or products, leading to profound changes in their catalytic properties. A critical diagnostic parameter for the prevalence of these interactions is the loss of homotropic and heterotropic effects on desensitization. The loss of allosteric sites results in marked changes in the conformation of the protein, catalytic properties and subunit interactions. The consequences of desensitization and methods employed are reviewed citing aspartale transcrbornylace, phosphofructokinase, phosphorylase, fructose biphosphatase, citrate synthase, nucleotide pyrophosphatase and other enzymes as examples. Desensitization of aspartate transcarbamylase by (i) dissociation using p-hydroxymercuribcnzoate (ii) causing local conformational changes with. low concentrations of urea: (iii) limited proteolysis
(iv) X-ray irradiation; (v) mutation ~ and (vi) constructing hybrid enzyme molecules composed of dlemically modified catalytic and regulatory subunits, clearly showed that the loss. of positive and negative heterotropic effects originated at the same location on the emzyme and were transmitted among the submits by similar mechanisms. These studies emphasized that there are no discrete cooperative units within the enzyme molecule but the allosteric transitions promoted by ligands are fully cooperative.

Simple manipulations such as dilution, changes in ionic strength, pH, storage conditions enzymatic methods like limited proteolysis, phosphorylation and dephosphorylatIon, adcnylatioli and deadenylation, interaction with dyes such as cibacron blue F3GA; chemical n10difications using general. group specific reagents or specific affinity labels; and association-dissociation, have been used to desensitize !he regulatory enzymes cited in the review. From such studies it can be concluded that binding per  allosteric site is not sufficient to evoke cooperativity, but this binding should be followed by a meaningful conformational change leading to' an alteration in the subunit interactions.