The tertiary origin of the allosteric activation of E. coli glucosamine-6-phosphate deaminase studied by sol-gel nanoencapsulation of its T conformer.

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 Kt and Kr 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

Reference values for the wild-type enzyme [20] and for the mutant F in solution.

a<p>Parameters obtained by fitting velocity against substrate concentration data to the MWC general equation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096536#pone.0096536-Henry1" target="_blank">[6]</a>.</p>b<p>Obtained by fitting velocity data to Hill equation.</p

Time-course of the tertiary conformational relaxation of the nanoencapsulated T-conformer, analyzed through the catalytic activity of EcGNPDA.

<p>(A) Plot of velocities against substrate concentrations at a fixed (0.3 mM) activator concentration. Substrate concentration was varied in an interval far below the <i>K</i>_t value for the enzyme in the T state (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096536#pone-0096536-t002" target="_blank">Table 2</a>), to mainly reveal the activity of the high affinity subunits. Data were fitted to hyperbola. The enzyme concentration was 2 nM. The symbols representing each incubation time are equal in the three panels. (B) Scatchard plot of the same data. Note the absence of homotropic cooperativity and the time-dependent recruitment of subunits in a high affinity state. The plotted lines were obtained by simulating the corresponding fitted transform. (C) Time-course of the allosteric activation of the T conformer, appreciated as an exponential increase of the apparent <i>V</i>_max. Data were fitted to the first order equation yielding a <i>k</i>_obs at 0.3 mM GlcNAc6P of 1.6×10⁻⁴±0.2×10⁻⁴ s⁻¹. Note that along the assay, the enzyme was already in contact with GlcNAc6P; for this reason the allosteric activation is in progress before starting the enzyme assay. Due to this experimental constraint, the observed span of fractional <i>V</i>_max values is lesser than one (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096536#pone-0096536-g002" target="_blank">Figure 2C</a>), even when referred to the extrapolated value at zero time.</p

Relaxation kinetics of the nanoencapsulated F mutant in T conformation: effect of GlcNAc6P concentration.

<p>The florescence decay curves recorded at different activator concentrations were fitted to a single exponential function and the obtained <i>k</i>_obs values were replotted as a function of the allosteric activator concentration. Data were fitted to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096536#pone.0096536.e002" target="_blank">equation 2</a>. The resulting rate constants are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096536#pone-0096536-t003" target="_blank">Table 3</a>. The inset shows an example of one of the recorded relaxation curves fitted to the first-order equation (dashed red line). This example corresponds to the highest GlcNAc6P concentration tested (1 mM) (red symbol).</p

Proposed two-step mechanism.

<p>The scheme depicts the mechanism with a fast step of activator binding, followed by a slow tertiary transition.</p

Circular Dichroism spectra of GNPDA-doped gels in the aromatic absorption range.

<p>(A) continuous line, CD spectrum of a monolith doped with ligand-free enzyme in T conformer; dashed line, spectrum of the same T gel equilibrated with 1 mM GlcNAc6P; dotted line, CD spectrum of a monolith doped with ligand-free enzyme in R conformer; dotted-dashed line, spectrum of the same R gel equilibrated with 1 mM GlcNAc6P. (B) Time course of CD change at 274 nm. The ligand-free enzyme nanoencapsulated in either quaternary conformer was added with 1 mM GlcNAc6P: (•), T form; (○), R form. The decay curve given by the R conformer was fitted to a simple exponential function. (C) Differential CD spectrum between the activator-saturated conformers and its correspondent ligand-free forms, showing the ellipticity change of the nanoencapsulated enzyme produced by GlcNAc6P binding. Red line: T_A-T₀; green line, R_A - R₀. (D) CD spectral difference between the activator-saturated R doped gel and the T₀ gel. These forms of the enzyme are equivalent to the extreme states observed in solution (T₀ and R_A); this differential spectrum matches with the corresponding spectral changes reported in solution <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096536#pone.0096536-Altamirano3" target="_blank">[34]</a>.</p

Kinetic constants for the deamination reaction of the nanoencapsulated T conformer of EcGNPDA.

a<p>From experiments shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096536#pone-0096536-g001" target="_blank">Figure 1</a>; parameters were obtained from global data fitting to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096536#pone.0096536.e001" target="_blank">equation 1</a>.</p>b<p>From experiment in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096536#pone-0096536-g002" target="_blank">Figure 2</a> that detects the catalytic activity of the high-affinity tertiary state. The <i>K</i>_r value is obtained from fitting to hyperbola.</p