A General Acid−Base Mechanism for the Stabilization of a Tetrahedral Adduct in a Serine−Carboxyl Peptidase:  A Computational Study

Wlodawer

et al. 2007

Biochemistry

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Quantum mechanical/molecular mechanical molecular dynamics and free energy simulations are performed to study the acylation reaction catalyzed by kumamolisin-As, a serine-carboxyl peptidase, and to elucidate the catalytic mechanism and the origin of substrate specificity. It is demonstrated that the nucleophilic attack by the serine residue on the substrate may not be the rate-limiting step for the acylation of the GPH*FF substrate. The present study also confirms the earlier suggestions that Asp164 acts as a general acid during the catalysis and that the electrostatic oxyanion hole interactions may not be sufficient to lead a stable tetrahedral intermediate along the reaction pathway. Moreover, Asp164 is found to act as a general base during the formation of the acyl-enzyme from the tetrahedral intermediate. The role of dynamic substrate assisted catalysis (DSAC) involving His at the P 1 site of the substrate is examined for the acylation reaction. It is demonstrated that the bond-breaking and -making events at each stage of the reaction trigger a change of the position for the His side chain and lead to the formation of the alternative hydrogen bonds. The back and forth movements of the His side chain between the CdO group of Pro at P 2 and O δ2 of Asp164 in a ping-pong-like mechanism and the formation of the alternative hydrogen bonds effectively lower the free energy barriers for both the nucleophilic attack and the acyl-enzyme formation and may therefore contribute to the relatively high activity of kumamolisin-As toward the substrates with His at the P 1 site.Kumamolsin-As belongs to the recently characterized family of serine-carboxyl peptidases (sedolisins) that were originally described by Murao, Oda, and co-workers about 20 years ago (1-3). Sedolisins are present in a wide variety of organisms, including archaea, bacteria, molds, slime molds (mixomycetes), amoebas, fishes, and mammals, and they are active at low pH and often high temperature. This family of enzymes seems to have been derived through divergent evolution from a common ancestor with classical serine peptidases; the structural studies (4-10) showed that sedolisins have a fold resembling that of subtilisin, although they are significantly larger (the mature catalytic domains contain approximately 375 amino acid residues). One interesting question is why nature needs another family of subtilisin-like peptidases. For bacterial sedolisins, one possible explanation is that some organisms might have to utilize such evolved serine-carboxyl peptidases for their survival, presumably under acidic environments and high temperature.The biological importance of sedolisins in mammals has already been demonstrated by the fact that mutations leading to the loss of the human enzyme CLN2 (11-13), another member of the sedolisin family, result in a fatal neurodegenerative disease, classical late-infantile neuronal ceroid lipofuscinosis (14).The three-dimensional structures are available for three members of the sedolisin family, including sedolisin...

supporting

confidence: 88%

Section: Structural and Dynamic Properties Of The Substratementioning

confidence: 99%

mentioning

confidence: 99%

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The QM/MM Molecular Dynamics and Free Energy Simulations of the Acylation Reaction Catalyzed by the Serine-Carboxyl Peptidase Kumamolisin-As

Wlodawer

et al. 2007

Biochemistry

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“…As discussed in our earlier papers, the corresponding methylation reactions are expected to be more efficient, as a part of the TS stabilization would be already reflected in the reactant complexes. [16][17][18][19][20][21] In contrast, for the methylation reactions involving N 1 , the reactant complexes (Figs. 3A and 7A) are significantly distorted from the corresponding TS structures (Figs.…”

Section: Discussionmentioning

confidence: 99%

“…The SCC-DFTB approach has been used previously on a number of systems by us, and the results seem to be reasonable. [16][17][18][19][20][21] We have compared the energetic results obtained from the SCC-DFTB and B3LYP/6-31G** calculations for the methyl transfer in a simple model system that resembles the methyl transfer in PRMTs (see Supplementary data). It was found that the potential energy function obtained from SCC-DFTB is quite similar to that obtained from the B3LYP/6-31G** calculations.…”

Section: Methodsmentioning

confidence: 99%

QM/MM MD and free energy simulations of the methylation reactions catalyzed by protein arginine methyltransferase PRMT3

Chu

2013

Can. J. Chem.

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Protein arginine N-methyltransferases (PRMTs) catalyze the transfer of methyl group(s) from S-adenosyl-L-methionine (AdoMet) to the guanidine group of arginine residue in abundant eukaryotic proteins. Two major types of PRMTs have been identified in mammalian cells. Type I PRMTs catalyze the formation of asymmetric -N G , N G -dimethylarginine (ADMA), while Type II PRMTs catalyze the formation of symmetric -N G , N= G -dimethylarginine (SDMA). The two different methylation products (ADMA or SDMA) of the substrate could lead to different biological consequences. Although PRMTs have been the subject of extensive experimental investigations, the origin of the product specificity remains unclear. In this study, quantum mechanical/ molecular mechanical (QM/MM) molecular dynamics (MD) and free energy simulations are performed to study the reaction mechanism for one of Type I PRMTs, PRMT3, and to gain insights into the energetic origin of its product specificity (ADMA). Our simulations have identified some important interactions and proton transfers involving the active site residues. These interactions and proton transfers seem to be responsible, at least in part, in making the N 2 atom of the substrate arginine the target of the both 1st and 2nd methylations, leading to the asymmetric dimethylation product. The simulations also suggest that the methyl transfer and proton transfer appear to be somehow concerted processes and that Glu326 is likely to function as the general base during the catalysis.Key words: protein arginine methyltransferases, product specificity, QM/MM, MD and free energy simulations, reaction mechanism.Résumé : Les protéines arginine N-méthyltransférases (PRMT) catalysent le transfert d'un ou de plusieurs groupes méthyle de la S-adénosyl-L-méthionine (AdoMet) au groupe guanidine du résidu arginine dans les protéines abondantes chez les eucaryotes. Deux types importants de PRMT ont été identifiés dans les cellules des mammifères. Les PRMT de type I catalysent la formation de -N G , N G -diméthylarginine asymétrique (DMAA), tandis que les PRMT de type II catalysent la formation de -N G , N= G -diméthylarginine symétrique (DMAS). Les conséquences biologiques de ces deux produits de méthylation distincts (DMAA ou DMAS) du substrat pourraient différer. Alors que les PRMT ont fait l'objet d'études expérimentales approfondies, l'origine de la spécificité du produit demeure obscure. Dans la présente étude, on procède à des simulations de dynamique moléculaire (DM) et d'énergies libres par des méthodes de mécanique quantique/mécanique moléculaire (MQ/MM) afin d'étudier le mécanisme de réaction pour l'une des PRMT de type I, la PMRT3, et de dégager des indices quant à l'origine énergétique de la spécificité de son produit (DMAA). Nos simulations ont permis d'identifier certaines interactions et certains transferts de proton importants faisant intervenir les résidus aux sites actifs. Ces interactions et transferts de protons semblent être responsables, du moins en partie, du fait que l'atome N 2 du...

Understanding the Autocatalytic Process of Pro‐kumamolisin Activation from Molecular Dynamics and Quantum Mechanical/Molecular Mechanical (QM/MM) Free‐Energy Simulations

Yao

Wlodawer

2013

Chemistry A European J

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