Figure 1.
Acylphosphatases use an invariant arginine residue to catalyze the hydrolysis of its substrates.
(A) The transition state of the enzyme-catalyzed hydrolysis of acylphosphate. (B) Schematic representation of the thermophilic PhAcP. The substrate acylphosphate was modeled to the active-site cradle, P-loop, by docking and molecular modeling [14]. The role of the active-site arginine residue (Arg-20) is to stabilize the negative charges in the transition state. In the structures of all thermophilic acylphosphatases determined to date, the active-site arginine residue forms a salt-bridge with the C-terminal carboxyl group.
Figure 2.
The salt-bridge restraining the active-site arginine residue resulted in a stronger temperature dependency of the acylphosphatase activity.
(A) The active-site salt-bridge (orange dotted line) between the guanido group of Arg-20 and the C-terminal carboxyl group of Gly-91 in PhWT (in yellow) is removed in PhG91A (in green). (B) Replacing the C-terminal residue of HuAcP with a glycine residue facilitates the formation of the active-site salt-bridge (orange dotted line) in HuG99 (in yellow). Such salt-bridge is absent in the pseudo-wild-type HuA99 (in green). (C) The Arrhenius plots for PhWT (open circle), PhG91A (open square), HuG99 (filled circle), and HuA99 (filled square). The data showed that the salt-bridge bearing acylphosphatases (PhWT and HuG99, solid line) had a steeper slope than the variants (PhG91A and HuA99, dotted line) lacking the salt-bridge.
Table 1.
Kinetics parameters of acylphosphatases with and without the active-site salt-bridge.
Figure 3.
Removal of the active-site salt-bridge decreases both activation enthalpy and entropy.
Changes in activation free energy (ΔΔG#, open circles, solid lines), activation enthalpy (ΔΔH#, dotted lines), and activation entropy (TΔΔS#, open diamond, solid lines) upon removal of the active-site salt-bridge were calculated as described in Table 1. As shown, removal of the salt-bridge leads to large negative values of both ΔΔH# and TΔΔS#, while the effect on activation free energy at ∼298 K was minimal due to enthalpy-entropy compensation.
Table 2.
Thermodynamics parameters for binding of substrate-analogue determined by isothermal titration calorimetry.
Figure 4.
The active-site salt-bridge restricts the side-chain conformational freedom of the active-site arginine residue.
The local flexibility of the active-site arginine residue (Arg-20 in PhWT and PhG91A or Arg-23 in HuG99 and HuA99) was examined by MD simulations. For acylphosphatases with the salt-bridge (PhWT and HuG99), the χ1, χ2, χ3, and χ4 dihedral angles of the arginine residue were confined to the values of ∼300°, ∼180°, ∼300°, and ∼180°, respectively. In other words, the side-chain of the arginine residue populates mainly in the mtm180° rotamer. According to the convention of Lovell et al. [18], “p,” “t,” and “m” refers to dihedral angles of 60°, 180°, and 300°, respectively. For acylphosphatases without the salt-bridge (PhG91A and HuA99), transitions from the mtm180° to other rotamer conformations (ptt180°, ttp180°, and mtt180°) were evident.
Figure 5.
The active-site arginine residue of acylphosphatase adopts the mtm180° rotamer conformation for catalysis.
The model of enzyme-transition-state complex was derived from the model of enzyme-substrate complex [14] by orientating the phosphorus atom towards the water molecule. Hydrogen bonds and salt-bridges are denoted by orange dotted lines. According to the proposed model, the guanido group of Arg-20 can form charge-charge interactions to stabilize the transition-state when the residue adopts the mtm180° rotamer conformation. For the mtt180°, ttp180°, and ptt180° rotamer conformations, the guanido group is too far away to form any favorable interactions with the transition state.