Nature has devised intrinsic electric fields (IEFs) that are engaged in electrostatic catalysis of enzymes. But, how does the IEF target its function in enzymes that involve several reaction steps in catalytic cycles? To decipher the impact of the IEF on the catalytic cycle of an enzyme system, we have performed molecular dynamics and quantum-mechanical/molecular-mechanical (QM/MM) simulations on tyrosine hydroxylase (TyrH). The catalytic cycle of TyrH involves two reaction stages: the activation of H2O2 to form the active species of compound I (Cpd I), in the first stage, and the Cpd I-mediated hydroxylation of l-tyrosine to l-DOPA, in the second stage. For the first stage, the QM/MM calculations show that a heme-propionate group functions as a base to catalyze the O–O heterolysis reaction. For the second stage, the study reveals that the reaction is initiated by the His88-mediated proton-coupled electron transfer followed by the oxygen atom transfer from compound II (Cpd II) to the l-Tyr substrate. Importantly, our calculations demonstrate that the IEF in TyrH is optimized to promote the O–O bond heterolysis that generates the active species of the enzyme, Cpd I. However, the same IEF slows down the subsequent aromatic hydroxylation. Thus, the IEF in the TyrH enzymes does not catalyze the product formation step, but will selectively boost one or more challenging steps in the catalytic cycle. These findings have general implications on O2/H2O2-dependent metalloenzymes, which can expand our understanding of how nature has used electric fields as “smart reagents” in modulating the catalytic reactivity.
The combined molecular dynamics (MD) simulations and quantum mechanical/molecular mechanics (QM/MM) calculations have been performed to address the longstanding issue of the "dioxygen activation" by the nonheme diiron monooxygenase myo-inositol oxygenase (MIOX). MIOX utilizes a mixed-valence Fe2(III)Fe1(II) cluster for catalysis. It is well recognized that the Fe2(III) site is responsible for the substrate myo-inositol (MI) binding, while the Fe1(II) site is responsible for O 2 binding and activation. However, it is enigmatic how the O−O bond of oxygen is reductively cleaved in the absence of additional reductants. In this study, we demonstrate a spin-regulated inner-sphere electron-transfer mechanism that is involved in the catalytic reactions of MIOX. Because of the Pauli principle and exchangeenhanced reactivity, the spin-regulated inner-sphere electron transfer enables the formation of an unprecedented Fe2(III)Fe1(II)-peroxyhemiketal intermediate that is responsible for the reductive O−O cleavage. In contrast to Fe1(III)-mediated O−O cleavage in the Fe2(II)Fe1(III)-peroxyhemiketal intermediate proposed previously, our calculations demonstrate that the proton transfer-triggered Fe1−O cleavage in Fe2(III)Fe1(II)-peroxyhemiketal intermediate is the most favorable pathway, leading to MI-OOH intermediate and the Fe1(II) species. The following Fe1(II)-mediated O−O homolysis in MI-OOH generates the substrate radical and Fe(III)−OH species, during which the Fe1(IV)O intermediate would be bypassed. Thus, our calculations show that both Fe sites are cooperately involved in O 2 activation in MIOX and such cooperation is well regulated by the spin-dependent innersphere electron transfer. These findings of O 2 activation by MIOX may have far-reaching implications on other related nonheme diiron monooxygenases.
Acid phosphatases (APases) are attractive enzymes for catalyzing large-scale industrial phosphorylation reactions owing to their capacity of utilizing cheap phosphate donors as phosphate sources as well as their broad substrate spectrum. However, APases exhibit strong hydrolytic activity that usually overwhelms the needed phosphorylation reaction. In the present study, we have solved the crystal structure of APase from Pseudomonas aeruginosa (PaAPase) and unraveled the mechanism of PaAPase-catalyzed L-ascorbic acid phosphorylation using multiscale computational studies. In addition, we have engineered the charged residues near the active site to investigate the local electric field effects on modulating the competition between hydrolysis and phosphorylation in PaAPase. In the optimal variant of Q6 containing Asp135 → Arg135 mutation, the corresponding phosphorylation/hydrolysis ratios have increased by 2.9-fold compared with those in the wild-type enzyme. In particular, our simulations show that the local electric field of Q6 could remarkably inhibit the hydrolysis of the phospho-His171 intermediate while having relatively minor effects on the overall phosphorylation reactions. Such an introduced local electric field shifts the phosphorylation/hydrolysis balance in favor of phosphorylation reaction. Our combined experiments and theories demonstrate that protein engineering focusing on local electric field optimization is a practical strategy for modulating enzymatic reactivity.
A solvated proton in water is often characterized as a charge or structural defect, and it is important to track its evolution on-the-fly in certain dynamics simulations. Previously, we introduced the proton indicator, a pseudo-atom, whose position approximates the location of the excess proton modeled as a structural defect. The proton indicator generally yields a smooth trajectory of a hydrated proton diffusing in aqueous solutions, including in the events of stepwise proton transfer via the Grotthuss mechanism; however, the proton indicator did not perform well in the events of concerted proton transfer, for which it occasionally yielded large position displacements between two successive time steps. To overcome this hurdle, we develop a new algorithm of a proton indicator with greatly enhanced performance for concerted proton transfer in bulk water. A protocol is proposed to exhaustively explore the hydrogen-bonding network of the water wires over which the excess proton is delocalized and to properly account for the contributions of the water molecules in this network as the geometry evolves. The new proton indicator (called Indicator 2.0) is assessed in dynamics simulations of an excess proton in bulk water and in specially constructed model systems of more complex architectures. The results demonstrate that the new indicator yields a smooth trajectory in both stepwise and concerted proton transfers.
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