On the Catalytic Activity of the Engineered Coiled-Coil Heptamer Mimicking the Hydrolase Enzymes: Insights from a Computational Study.

Mario Prejanò,Tiziana Marino,Isabella Romeo,Nino Russo

doi:10.3390/ijms21124551

Mario Prejanò, Tiziana Marino + Show 2 more

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https://doi.org/10.3390/ijms21124551

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Abstract

Recently major advances were gained on the designed proteins aimed to generate biomolecular mimics of proteases. Although such enzyme-like catalysts must still suffer refinements for improving the catalytic activity, at the moment, they represent a good example of artificial enzymes to be tested in different fields. Herein, a de novo designed homo-heptameric peptide assembly (CC-Hept) where the esterase activity towards p-nitro-phenylacetate was obtained for introduction of the catalytic triad (Cys-His-Glu) into the hydrophobic matrix, is the object of the present combined molecular dynamics and quantum mechanics/molecular mechanics investigation. Constant pH Molecular Dynamics simulations on the apoform of CC-Hept suggested that the Cys residues are present in the protonated form. Molecular dynamics (MD) simulations of the enzyme–substrate complex evidenced the attitude of the enzyme-like system to retain water molecules, necessary in the hydrolytic reaction, in correspondence of the active site, represented by the Cys-His-Glu triad on each of the seven chains, without significant structural perturbations. A detailed reaction mechanism of esterase activity of CC-Hept-Cys-His-Glu was investigated on the basis of the quantum mechanics/molecular mechanics calculations employing a large quantum mechanical (QM) region of the active site. The proposed mechanism is consistent with available esterases kinetics and structural data. The roles of the active site residues were also evaluated. The deacylation phase emerged as the rate-determining step, in agreement with esterase activity of other natural proteases.

Highlights

Proteases are a family of enzymes whose function is to catalyze the hydrolysis of peptide bonds
Constant pH Molecular Dynamics (CpHMD) (Figure 1) emerged a configuration with six His protonated in Nδ and one in Nε
We have undertaken a detailed study based on both Molecular dynamics (MD) simulations and quantum mechanical (QM)/MM calculations to investigate the ester hydrolysis promoted by a totally engineered protein converted into hydrolase enzyme for installation of the common Cys-His-Glu triad in cysteine proteases, on every chain of the coiled-coil heptamer

Summary

Introduction

Proteases are a family of enzymes whose function is to catalyze the hydrolysis of peptide bonds. Physiologically these enzymes are peptide bond hydrolases, they can promote the hydrolysis of substrates of different natures such as esters, thioesters, anhydrides, and acid chlorides. Despite the differences in structure, active site, and metal center present in the four main classes of proteases (serine proteases, cysteine proteases, aspartyl proteases, and metalloproteases), all of them have in common the use of a nucleophile whose activation can occur in different ways. Previous studies in mechanistic enzymology, choosing the chymotrypsin as a prototype for a variety of enzyme classes including proteases, allowed to evidence the peptide hydrolysis occurring in different steps (activation, acylation, and de-acylation). The activated nucleophile attacks the carbon of the peptide bond and the electrons in the carbon-oxygen double bond migrate onto the oxygen

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