Abstract
The role of tertiary conformational changes associated to ligand binding was explored using the allosteric enzyme glucosamine-6-phosphate (GlcN6P) deaminase from Escherichia coli (EcGNPDA) as an experimental model. This is an enzyme of amino sugar catabolism that deaminates GlcN6P, giving fructose 6-phosphate and ammonia, and is allosterically activated by N-acetylglucosamine 6-phosphate (GlcNAc6P). We resorted to the nanoencapsulation of this enzyme in wet silica sol-gels for studying the role of intrasubunit local mobility in its allosteric activation under the suppression of quaternary transition. The gel-trapped enzyme lost its characteristic homotropic cooperativity while keeping its catalytic properties and the allosteric activation by GlcNAc6P. The nanoencapsulation keeps the enzyme in the T quaternary conformation, making possible the study of its allosteric activation under a condition that is not possible to attain in a soluble phase. The involved local transition was slowed down by nanoencapsulation, thus easing the fluorometric analysis of its relaxation kinetics, which revealed an induced-fit mechanism. The absence of cooperativity produced allosterically activated transitory states displaying velocity against substrate concentration curves with apparent negative cooperativity, due to the simultaneous presence of subunits with different substrate affinities. Reaction kinetics experiments performed at different tertiary conformational relaxation times also reveal the sequential nature of the allosteric activation. We assumed as a minimal model the existence of two tertiary states, t and r, of low and high affinity, respectively, for the substrate and the activator. By fitting the velocity-substrate curves as a linear combination of two hyperbolic functions with K t and K r as KM values, we obtained comparable values to those reported for the quaternary conformers in solution fitted to MWC model. These results are discussed in the background of the known crystallographic structures of T and R EcGNPDA conformers. These results are consistent with the postulates of the Tertiary Two-States (TTS) model.
Highlights
The concept of allostery, introduced by Monod et al in 1963, embodies one of the fundamental principles of life [1]
We characterized the allosteric activation of the nanoencapsulated T conformer of EcGNPDA under conditions of total suppression of the quaternary transition
Activation by GlcNAc6P is elicited by its binding to face A of each subunit (Figure 6), which undergoes a conformational change
Summary
The concept of allostery, introduced by Monod et al in 1963, embodies one of the fundamental principles of life [1]. Monod et al in their landmark model presented a unified theory of cooperativity and heterotropic allosteric effects [3] Their model conceived allosteric transitions as symmetric quaternary rearrangements of subunits between two extreme conformational states T and R, with different binding affinities (MWC model [3]). The evidence that the tertiary conformational changes alone can produce heterotropic, that is, allosteric effects in hemoglobin contradict the original postulates of the MWC model [5] From these and other experimental evidences, Henry et al proposed a MWC-inspired theory to account for the role of tertiary allosteric transitions and its coupling to the quaternary transition in hemoglobin [6]. The tertiary-quaternary coupling is explained by the mutual biasing between the tertiary and quaternary conformational states, which undergo sequential and concerted transitions respectively
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