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. 2009 May 1;284(18):12136-44.
doi: 10.1074/jbc.M806228200. Epub 2009 Feb 26.

Covalently bound substrate at the regulatory site of yeast pyruvate decarboxylases triggers allosteric enzyme activation

Steffen Kutter  1 , Manfred S Weiss, Georg Wille, Ralph Golbik, Michael Spinka, Stephan König
Affiliations

Covalently bound substrate at the regulatory site of yeast pyruvate decarboxylases triggers allosteric enzyme activation

Steffen Kutter et al. J Biol Chem. .

Abstract

The mechanism by which the enzyme pyruvate decarboxylase from two yeast species is activated allosterically has been elucidated. A total of seven three-dimensional structures of the enzyme, of enzyme variants, or of enzyme complexes from two yeast species, three of them reported here for the first time, provide detailed atomic resolution snapshots along the activation coordinate. The prime event is the covalent binding of the substrate pyruvate to the side chain of cysteine 221, thus forming a thiohemiketal. This reaction causes the shift of a neighboring amino acid, which eventually leads to the rigidification of two otherwise flexible loops, one of which provides two histidine residues necessary to complete the enzymatically competent active site architecture. The structural data are complemented and supported by kinetic investigations and binding studies, providing a consistent picture of the structural changes occurring upon enzyme activation.

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Figures

SCHEME 1.
SCHEME 1.
Chemical structures of the substrate pyruvate, the activators pyruvamide and methyl acetylphosphonate, and the thiohemiketal from pyruvate and cysteine, respectively.
FIGURE 1.
FIGURE 1.
MAP kinetics. A, influence of MAP on the shape of the v versus [S] plot (circles, 0 mm MAP, squares, 20 mm MAP, triangles, 40 mm MAP, and inverse triangles, 75 mm MAP). The lines represent sigmoid fits according to equation v([S]) = Vmax · [S]2/(A + B · [S] + [S]2) with the following values for the parameters for Vmax (units/mg), A (mm2), and B (mm), respectively, 0 mm MAP, 8.77, 2.52, 0.27; 20 mm MAP, 9.16, 0.72, 1.88; 40 mm MAP, 9.30, 10-9, 2.83; 75 mm MAP, 9.39, 10-9, 3.26. The uncertainties of these values are less than 20% throughout, except for the A values in the presence of 40–75 mm MAP, which are essentially zero, demonstrating Michaelis-Menten behavior of the activated enzyme with B as apparent Km. Inset, enlarged view at low substrate concentrations. B, transients of the KlPDC-catalyzed reaction at 3 mm PYR after preincubation with MAP (increasing concentrations from top to bottom as in A). For better comparison, original data are normalized to the steady state rate and to the same initial absorbance. C, dependence of the ratio of initial rate (v0) and steady state rate (vSS) on MAP concentration. The rates were obtained from transients (see B for primary data) according to Equation 2. The line represents a fit to the equation v0/vSS = K1 [L] + [L]2/(A + K1 · [L] + [L]2) with A = KL · Kilo · K1. The dissociation constants correspond to Scheme 2, and [L] represents the MAP concentration during preincubation. Fit parameters are K1 = 2.5 ± 2 mm, A = 1460 ± 142 mm2. D, activator concentration dependence of the apparent activation rate constant kobs (circles, PYR; squares, MAP). The kobs values were obtained from discontinuous measurements (for details, see the section “Kinetic Measurements”). The error bars represent the fitting errors. In the absence of MAP, the line represents the fit according to the equation kobs = kiso · [S]/([S] + Ka) + k-iso · KmS/(KmS + [S]) (16); in the presence of MAP, a line is drawn for better visualization only.
SCHEME 2.
SCHEME 2.
Proposed kinetic model for the allosteric activation of yeast pyruvate decarboxylases in simultaneous presence of both the substrate S and the artificial activator L. Ka and KL are the primary dissociation constants at the regulatory site for S and L, respectively. kiso and kilo represent the forward rate constants for the conformation change driven by S and L, respectively. k-iso and k-ilo are the associated rate constants for the reverse reaction. Kiso and Kilo are defined by Kiso = (k-iso/kiso) and Kilo = (k-ilo/kilo), respectively. KmS is the Michaelis-Menten constant for the substrate-activated enzyme, whereas Kml refers to the ligand-activated enzyme. K1 is the dissociation constant of the artificial activator for the ligand-activated enzyme. Ligands/substrates written on the left of the enzyme species are bound to the regulatory site of the enzyme, and those written on the right are bound to the active site. In the absence of L, this activation scheme reduces to the established model of reference (16).
FIGURE 2.
FIGURE 2.
Dependence of the scattering parameter RG of PDC on the concentration of the added activator. In the case of PA and MAP, KlPDC was used, and in the case of PYR, ScPDCE477Q was used (triangles, PA; squares, MAP; inverse triangles, PYR; lines, hyperbolic fits for KlPDC, sigmoid fit for ScPDCE477Q). The following values for half-saturation were obtained: for PYR, 5.7 ± 0.23 mm (n = 1.85); for MAP, 20.7 ± 2.39 mm; for PA, 37.8 ± 1.36 mm).
FIGURE 3.
FIGURE 3.
The overall crystal structures of PDC tetramers representing MAP-KlPDC and PYR-ScPDC variants in A and of native KlPDC (28), PA-ScPDC (38), and ketomalonate-activated ScPDC (39) in B. The subunits are shown with different colors in space-filling mode.
FIGURE 4.
FIGURE 4.
View of the regulatory sites with the activators bound covalently to residue Cys-221 for MAP-KlPDC (A) and PYR-ScPDCE477Q (B). Electron density is shown at a σ-level of 2.0 in the 2Fo - Fc map (blue) and at a σ-level of 4.0 in the omit map (green). Amino acid residues are illustrated in stick mode. The labeled residues can directly interact with the thiohemiketal. Labels in A correspond also to B.
FIGURE 5.
FIGURE 5.
View from the surface of KlPDC molecule down to one active site. A and B, the location of the histidine residues 114 and 115 in native KlPDC (A)- and MAP-activated KlPDC (B). The cofactor ThDP and the histidine residues are presented in stick mode (individually colored atoms), and all other residues are shown as a tube of the Cα line, with loop 104–113 in blue, loop 288–304 in red, and the C-terminal helix in green.
FIGURE 6.
FIGURE 6.
Snapshots of the signal transduction pathway from the regulatory to the active site in yeast PDCs. The different subunits are shown as Cα trace in different colors, the labeled residues are shown as sticks, and the color of the label is that of the subunit the residue belongs to. A, overlay of one regulatory site before (carbon atoms in green) and after covalent binding of the activator MAP at Cys-221 (carbon atoms in beige). The 4 Å shift of the thiohemiketal is clearly to be seen. B, the shift of the thiohemiketal causes direct interactions to residues Ala-286 and Gly-287 and restructures the whole loop region 288–304 (beige tube; loop conformation before activation is shown in green). C, the structured loop 288–304 induces direct interactions to the other loop region 104–113, and the H-bonds between both loops are illustrated as blue dashed lines. The new conformation of this loop (beige, loop conformation before activation in green) reorients the adjacent residues His-114 and His-115, which now can directly interact with the activator molecule bound at the C2 atom of ThDP.
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