Substrate Inhibition by the Blockage of Product Release and Its Control by Tunnel Engineering

Kokkonen, Piia; Beier, Andy; Mazurenko, Stanislav; Damborský, Jiřı́; Bednář, David; Prokop, Zbyněk

doi:10.26434/chemrxiv.13311614

Cited by 7 publications

(8 citation statements)

References 34 publications

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“…Substrate inhibition is the most common deviation from Michaelis-Menten kinetics, occurring in about 25% of known enzymes. It is generally attributed to the formation of an unproductive enzyme-substrate complex after the simultaneous binding of two or more substrate molecules to the active site [37]. Thus, our model behaves much as in the case of a substrate inhibition mechanism, with one important exception: in our case, we have two enzyme monomers bound to one substrate molecule, whereas the substrate inhibition model considers the ES 2 system, i .…”

Section: Discussionmentioning

confidence: 99%

SARS-CoV-2 Main Protease: a Kinetic Approach

Rebetez¹

2022

Preprint

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In this article, I present a new model of the interaction of the main protease (Mpro) from SARS-CoV-2 virus with its substrate. The reaction scheme used to describe this mechanism is an extension of the well-known Michaelis Menten model proposed in 1913 by Leonor Michaelis and Maud Menten [1]. The model I present here takes into account that one Mpro enzyme monomer interacts with another Mpro monomer in the presence of the substrate, leading to the formation of an enzyme dimer bound to one substrate molecule. Indeed, this dimer is formed by the sequentially binding of one Mpro enzyme monomer to one molecule of substrate, followed by another Mpro enzyme monomer binding to this Mpro-substrate complex. This reaction mechanism is also known in the literature as substrate-induced dimerization [3]. Starting from this new reaction scheme established for this catalytic mechanism, I derived a mathematical expression describing the catalytic rate of the active Mpro enzyme dimer as a function of the substrate concentration [S]. The plot corresponding to this substrate-induced dimerization reaction shows a function f([S]) that is not monotonic, i.e. not strictly increasing or decreasing, but with a second derivative initially negative and then becoming positive after having passed the Vmax point. This is typically a type of curve showing a phenomenon like the one of substrate inhibition (for instance, inhibition by excess-substrate [7]). The graphical representation of this process shows an interesting behaviour: from zero micro M/s, the reaction rate increases progressively, similar to the kind of curve described by the Michaelis-Menten model. However, after having reached its maximum catalytic rate, Vmax, the reaction rate decreases progressively as we continue to increase the substrate concentration. I propose an explanation to this interesting behavior. At the moment where Vcat is maximum, we can assume that, in theory, every single substrate molecule in solution is bound to two enzyme monomers (i.e. to one active dimer). The catalytic rate is thus theoretically maximized. At the time where the reaction rate begins to decrease, we observe a new phenomenon that appears: the enzyme monomers begin to be "diluted" in the solution containing the excess substrate. The dimers begin to dissociate and to bind increasingly to the substrate as inactive monomers instead of active dimers. Hence, it is more and more unlikely for the enzyme monomers to sequentially bind twice to the same substrate molecule (here, [E] << [S]). Thus, at this stage, the substrate-induced dimerization occurs less often. At the limit, when the substrate is in high excess, there is virtually no more dimerization which occurs. This is one example of excess-substrate inhibition. Furthermore, after having established this fact, I wanted to see if this catalytic behavior was also observed in vitro. Therefore, I conducted an experiment where I measured the catalytic rate of the Mpro dimer for different substrate concentrations. The properties of my substrate construct were such, that I could determine the catalytic rate of the enzyme dimer by directly measuring the spectrophotometric absorbance of the cleaved substrate at lambda = 405 nm. The results show explicitly - within a margin of error - that the overall shape of the experimental curve looks like the one of the theoretical curve. I thus conclude that the biochemical behavior of the Mpro in vitro follows a new path when it is in contact with its substrate: an excess substrate concentration decreases the activity of the enzyme by the phenomenon of a type of excess-substrate inhibition. This finding could open a new door in the discovery of drugs directed against the Mpro enzyme of the SARS-CoV-2 virus, acting on the inhibition by excess-substrate of the Mpro enzyme, this protein being a key component in the metabolism of the virus. Furthermore, I have established that the maximum of the fitted curve, Vmax, depends only on [E]T and not on [S]. [S]_Vmax exhibits the same dependence pattern. Therefore, if I keep [E]T close to zero, the catalytic rate of the enzyme will also be greatly reduced, which can be understood intuitively. Finally, if we dilute the enzyme sufficiently in the host cell by injecting a suitably high concentration of the octapeptide substrate AVLQSGFR (an inhibitor of the original substrate), this artificial substrate will bind to the "intermediate" dimer from the polypeptide and prevent the precursor Mpro from auto-cleaving and dimerizing due to the "distorted key" effect of the octapeptide on the "intermediate" dimer. The precursor peptide Mpro will auto-cleave to a lesser extent than in the absence of the artificial octapeptide and thus the concentration of the total enzyme [E]T will be lowered in the cell. It would therefore be possible to control the virulence of the virus by adjusting the concentration of the artificial inhibitory octapeptide. However, this is only speculation and has yet to be verified in practice.

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Section: Discussionmentioning

confidence: 99%

SARS-CoV-2 Main Protease: a Kinetic Approach

Rebetez¹

2022

Preprint

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“…40 Considering that both sites in PchP are found in a common cavity, where the entrance to the AS implies a first access to the IS, the mechanism could be of the blockage type for the entry of substrates or for the release of products, such as the one recently proposed for the haloalkane dehalogenase of Sphingobium japonicum UT26. 41 However, despite still not knowing the biological meaning of PchP inhibition, it seems clear that because it is a differential and distinctive aspect with respect to PHOSPHO1 it can be considered for pharmacological purposes.…”

Section: ■ Discussionmentioning

confidence: 99%

Structural Insights into the Inhibition Site in the Phosphorylcholine Phosphatase Enzyme of Pseudomonas aeruginosa

Bustos

Hernández-Rodríguez

Poblete

et al. 2022

J. Chem. Inf. Model.

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Pseudomonas aeruginosa is a highly pathogenic Gram-negative microorganism associated with high mortality levels in burned or immunosuppressed patients or individuals affected by cystic fibrosis. Studies support a colonization mechanism whereby P. aeruginosa can breakdown the host cell membrane phospholipids through the sequential action of two enzymes: (I) hemolytic phospholipase C acting upon phosphatidylcholine or sphingomyelin to produce phosphorylcholine (Pcho) and (II) phosphorylcholine phosphatase (PchP) that hydrolyzes Pcho to generate choline and inorganic phosphate. This coordinated action provides the bacteria with carbon, nitrogen, and inorganic phosphate to support growth. Furthermore, PchP exhibits a distinctive inhibition mechanism by high substrate concentration. Here, we combine kinetic assays and computational approaches such as molecular docking, molecular dynamics, and free-energy calculations to describe the inhibitory site of PchP, which shares specific residues with the enzyme's active site. Our study provides insights into a coupled inhibition mechanism by the substrate, allowing us to postulate that the integrity of the inhibition site is needed to the correct functioning of the active site. Our results allow us to gain a better understanding of PchP function and provide the basis for a rational drug design that might contribute to the treatment of infections caused by this important opportunistic pathogen.

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“…Previous studies indicated that the characteristics of substrate access tunnels can have a decisive influence on enzyme-substrate specificity and activity [53][54][55][56]. In t-PlaF, we focused on T1 and T2 because only these two allow direct access of GPL or LGPL substrates from the membrane in the t-PlaF A configuration.…”

Section: Discussionmentioning

confidence: 99%

Substrate access mechanism in a novel membrane-bound phospholipase A of Pseudomonas aeruginosa concordant with specificity and regioselectivity

Ahmad

Strunk

Schott‐Verdugo

et al. 2021

Preprint

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PlaF is a cytoplasmic membrane-bound phospholipase A1 from Pseudomonas aeruginosa that alters the membrane glycerophospholipid (GPL) composition and fosters the virulence of this human pathogen. PlaF activity is regulated by a dimer-to-monomer transition followed by tilting of the monomer in the membrane. However, how substrates reach the active site and how the characteristics of the active site tunnels determine the activity, specificity, and regioselectivity of PlaF for natural GPL substrates has remained elusive. Here, we combined unbiased and biased all-atom molecular dynamics (MD) simulations and configurational free energy computations to identify access pathways of GPL substrates to the catalytic center of PlaF. Our results map out a distinct tunnel through which substrates access the catalytic center. PlaF variants with bulky tryptophan residues in this tunnel revealed decreased catalysis rates due to tunnel blockage. The MD simulations suggest that GPLs preferably enter the active site with the sn-1 acyl chain first, which agrees with the experimentally demonstrated PLA1 activity of PlaF. We propose that the acyl chain-length specificity of PlaF is determined by the structural features of the access tunnel, which results in favorable free energy of binding of medium-chain GPLs. The suggested egress route conveys fatty acid products to the dimerization interface and, thus, contributes to understanding the product feedback regulation of PlaF by fatty acid-triggered dimerization. These findings open up opportunities for developing potential PlaF inhibitors, which may act as antibiotics against P. aeruginosa.

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Substrate Inhibition by the Blockage of Product Release and Its Control by Tunnel Engineering

Cited by 7 publications

References 34 publications

SARS-CoV-2 Main Protease: a Kinetic Approach

SARS-CoV-2 Main Protease: a Kinetic Approach

Structural Insights into the Inhibition Site in the Phosphorylcholine Phosphatase Enzyme of Pseudomonas aeruginosa

Substrate access mechanism in a novel membrane-bound phospholipase A of Pseudomonas aeruginosa concordant with specificity and regioselectivity

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