A rigidifying salt-bridge favors the activity of thermophilic enzyme at high temperatures at the expense of low-temperature activity

Abstract

Background: Thermophilic enzymes are often less active than their mesophilic homologues at low temperatures. One hypothesis to explain this observation is that the extra stabilizing interactions increase the rigidity of thermophilic enzymes and hence reduce their activity. Here we employed a thermophilic acylphosphatase from Pyrococcus horikoshii and its homologous mesophilic acylphosphatase from human as a model to study how local rigidity of an active-site residue affects the enzymatic activity.

Methods and findings: Acylphosphatases have a unique structural feature that its conserved active-site arginine residue forms a salt-bridge with the C-terminal carboxyl group only in thermophilic acylphosphatases, but not in mesophilic acylphosphatases. We perturbed the local rigidity of this active-site residue by removing the salt-bridge in the thermophilic acylphosphatase and by introducing the salt-bridge in the mesophilic homologue. The mutagenesis design was confirmed by x-ray crystallography. Removing the salt-bridge in the thermophilic enzyme lowered the activation energy that decreased the activation enthalpy and entropy. Conversely, the introduction of the salt-bridge to the mesophilic homologue increased the activation energy and resulted in increases in both activation enthalpy and entropy. Revealed by molecular dynamics simulations, the unrestrained arginine residue can populate more rotamer conformations, and the loss of this conformational freedom upon the formation of transition state justified the observed reduction in activation entropy.

Conclusions: Our results support the conclusion that restricting the active-site flexibility entropically favors the enzymatic activity at high temperatures. However, the accompanying enthalpy-entropy compensation leads to a stronger temperature-dependency of the enzymatic activity, which explains the less active nature of the thermophilic enzymes at low temperatures.

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 . 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.

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.

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. , “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 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.