Properties of the histidine residues of indole-chymotrypsin. Implications for the activation process and catalytic mechanism.
Ontology highlight
ABSTRACT:
SUBMITTER: Cruickshank WH
PROVIDER: S-EPMC1165466 | biostudies-other | 1975 Jun
REPOSITORIES: biostudies-other
Similar Datasets
Project description:3-Chymotrypsin-like protease (3CLpro) is a promising drug target for coronavirus disease 2019 and related coronavirus diseases because of the essential role of this protease in processing viral polyproteins after infection. Understanding the detailed catalytic mechanism of 3CLpro is essential for designing effective inhibitors of infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Molecular dynamics studies have suggested pH-dependent conformational changes of 3CLpro, but experimental pH profiles of SARS-CoV-2 3CLpro and analyses of the conserved active-site histidine residues have not been reported. In this work, pH-dependence studies of the kinetic parameters of SARS-CoV-2 3CLpro revealed a bell-shaped pH profile with 2 pKa values (6.9 ± 0.1 and 9.4 ± 0.1) attributable to ionization of the catalytic dyad His41 and Cys145, respectively. Our investigation of the roles of conserved active-site histidines showed that different amino acid substitutions of His163 produced inactive enzymes, indicating a key role of His163 in maintaining catalytically active SARS-CoV-2 3CLpro. By contrast, the H164A and H172A mutants retained 75% and 26% of the activity of WT, respectively. The alternative amino acid substitutions H172K and H172R did not recover the enzymatic activity, whereas H172Y restored activity to a level similar to that of the WT enzyme. The pH profiles of H164A, H172A, and H172Y were similar to those of the WT enzyme, with comparable pKa values for the catalytic dyad. Taken together, the experimental data support a general base mechanism of SARS-CoV-2 3CLpro and indicate that the neutral states of the catalytic dyad and active-site histidine residues are required for maximum enzyme activity.
| S-EPMC9733303 | biostudies-literature
Project description:Elongation factor Tu (EF-Tu) is central to prokaryotic protein synthesis as it has the role of delivering amino-acylated tRNAs to the ribosome. Release of EF-Tu, after correct binding of the EF-Tu:aa-tRNA complex to the ribosome, is initiated by GTP hydrolysis. This reaction, whose mechanism is uncertain, is catalyzed by EF-Tu, but requires activation by the ribosome. There have been a number of mechanistic proposals, including those spurred by a recent X-ray crystallographic analysis of a ribosome:EF-Tu:aa-tRNA:GTP-analog complex. In this work, we have investigated these and alternative hypotheses, using high-level quantum chemical/molecular mechanical simulations for the wild-type protein and its His85Gln mutant. For both proteins, we find previously unsuggested mechanisms as being preferred, in which residue 85, either His or Gln, directly assists in the reaction. Analysis shows that the RNA has a minor catalytic effect in the wild-type reaction, but plays a significant role in the mutant by greatly stabilizing the reaction's transition state. Given the similarity between EF-Tu and other members of the translational G-protein family, it is likely that these mechanisms of ribosome-activated GTP hydrolysis are pertinent to all of these proteins.
| S-EPMC3753929 | biostudies-literature
Project description:The properties of a derivative of alpha-chymotrypsin in which histidine-57 has been methylated have been examined. Although the modified enzyme binds substrate with the same affinity as does native alpha-chymotrypsin, acylation and deacylation occur at much decreased rates. As for native alpha-chymotrypsin, a basic group of pK(a) approx. 7 is involved in both acylation and deacylation. The significance of these results is considered in relation to the normal function of histidine-57.
| S-EPMC1177107 | biostudies-other
Project description:Coronaviruses are responsible for multiple pandemics and millions of deaths globally, including the current pandemic of coronavirus disease 2019 (COVID-19). Development of antivirals against coronaviruses, including the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) responsible for COVID-19, is essential for containing the current and future coronavirus outbreaks. SARS-CoV-2 proteases represent important targets for the development of antivirals because of their role in the processing of viral polyproteins. 3-Chymotrypsin-like protease (3CLpro) is one such protease. The cleavage of SARS-CoV-2 polyproteins by 3CLpro is facilitated by a Cys145-His41 catalytic dyad. We here characterized the catalytic roles of the cysteine-histidine pair for improved understanding of the 3CLpro reaction mechanism, to inform the development of more effective antivirals against Sars-CoV-2. The catalytic dyad residues were substituted by site-directed mutagenesis. All substitutions tested (H41A, H41D, H41E, C145A, and C145S) resulted in a complete inactivation of 3CLpro, even when amino acids with a similar catalytic function to that of the original residues were used. The integrity of the structural fold of enzyme variants was investigated by circular dichroism spectroscopy to test if the catalytic inactivation of 3CLpro was caused by gross changes in the enzyme secondary structure. C145A, but not the other substitutions, shifted the oligomeric state of the enzyme from dimeric to a higher oligomeric state. Finally, the thermodynamic stability of 3CLpro H41A, H41D, and C145S variants was reduced relative the wild-type enzyme, with a similar stability of the H41E and C145A variants. Collectively, the above observations confirm the roles of His41 and Cys145 in the catalytic activity and the overall conformational fold of 3CLpro SARS-CoV-2. We conclude that the cysteine-histidine pair should be targeted for inhibition of 3CLpro and development of antiviral against COVID-19 and coronaviruses.
| S-EPMC8264439 | biostudies-literature
Project description:Histidine kinases (HK) are one of the main prokaryotic signaling systems. Two structurally conserved catalytic domains inside the HK enable autokinase, phosphotransfer, and phosphatase activities. Here, we focus on a detailed mechanistic understanding of the functional cycle of the WalK HK by a multi-scale simulation approach, consisting of classical as well as hybrid QM/MM molecular dynamics simulation. Strikingly, a conformational transition induced solely in DHp leads to the correct activated conformation in CA crucial for autophosphorylation. This finding explains how variable sensor domains induce the transition from inactive to active state. The subsequent autophosphorylation inside DHp proceeds via a penta-coordinated transition state to a protonated phosphohistidine intermediate. This intermediate is consequently deprotonated by a suitable nearby base. The reaction energetics are controlled by the final proton acceptor and presence of a magnesium cation. The slow rates of the process result from the high energy barrier of the conformational transition between inactive and active states. The phosphorylation step exhibits a lower barrier and down-the-hill energetics. Thus, our work suggests a detailed mechanistic model for HK autophosphorylation.
| S-EPMC11371814 | biostudies-literature
Project description:Tyrosinase is a copper-binding enzyme involved in melanin biosynthesis. However, the detailed structure of human tyrosinase has not yet been solved, along with the identification of the key sites responsible for its catalytic activity. We used site-directed mutagenesis to identify the residues critical for the copper binding of human tyrosinase. Seven histidine mutants in the two copper-binding sites were generated, and catalytic activities were characterised. The tyrosine hydroxylase activities of the CuA site mutants were approximately 50% lower than those of the wild-type tyrosinase, while the dopa oxidation activities of the mutants were not significantly different from that of wild-type tyrosinase. By contrast, mutations at CuB significantly decreased both tyrosine hydroxylation and dopa oxidation activities, confirming that the catalytic sites for these two activities are at least partially distinct. These findings provide a useful resource for further structural determination and development of tyrosinase inhibitors in the cosmetic and pharmaceutical industries.
| S-EPMC7144311 | biostudies-literature
Project description:BackgroundIdentifying the active site of an enzyme is a crucial step in functional studies. While protein sequences and structures can be experimentally characterized, determining which residues build up an active site is not a straightforward process. In the present study a new method for the detection of protein active sites is introduced. This method uses local network descriptors derived from protein three-dimensional structures to determine whether a residue is part of an active site. It thus does not involve any sequence alignment or structure similarity to other proteins. A scoring function is elaborated over a set of more than 220 proteins having different structures and functions, in order to detect protein catalytic sites with a high precision, i.e. with a minimal rate of false positives.ResultsThe scoring function was based on the counts of first-neighbours on side-chain contacts, third-neighbours and residue type. Precision of the detection using this function was 28.1%, which represents a more than three-fold increase compared to combining closeness centrality with residue surface accessibility, a function which was proposed in recent years. The performance of the scoring function was also analysed into detail over a smaller set of eight proteins. For the detection of 'functional' residues, which were involved either directly in catalytic activity or in the binding of substrates, precision reached a value of 72.7% on this second set. These results suggested that our scoring function was effective at detecting not only catalytic residues, but also any residue that is part of the functional site of a protein.ConclusionAs having been validated on the majority of known structural families, this method should prove useful for the detection of active sites in any protein with unknown function, and for direct application to the design of site-directed mutagenesis experiments.
| S-EPMC2632678 | biostudies-literature
Project description:The catalytic cycle for the serine protease ?-chymotrypsin was investigated in an attempt to determine the suitability of using the semiempirical method PM7 in the program MOPAC for investigating enzyme-catalyzed reactions. All six classical intermediates were modeled using standard methods, and were characterized as stable minima on the potential energy surface. Using a modified saddle point optimization method, five transition states were located and verified both by vibrational and by intrinsic reaction coordinate analysis. Some individual features, such as the hydrogen bonds in the oxyanion hole, the nature of various electrostatic interactions, and the role of Met192, were examined. This involved designing and running computational experiments to model mutations that would allow features of interest, in particular the energies involved, to be isolated. Three features within the enzyme were examined in detail: the reaction site itself, where covalent bonds were made and broken, the electrostatic effects of the buried aspartate anion, a passive but essential component of the catalytic triad, and the oxyanion hole, where hydrogen bonds help stabilize charged intermediates. With one minor exception, all phenomena investigated agreed with previously-reported descriptions. This result, along with the fact that all the techniques used were relatively straightforward, leads to the recommendation that PM7 and related methods, such as PM6-D3H4, are appropriate for modeling similar enzyme-catalyzed reactions. Graphical abstract Fifth of six transition states, showing water splitting into hydroxyl anion and a proton, to form the second tetrahedral intermediate and histidinium ion. Atoms of the water molecule involved in the hydrolysis are indicated by halos.
| S-EPMC5380709 | biostudies-literature
Project description:"Channel-like" self-assembled monolayers having aliphatic and aromatic diimines (denoted as Si@1DIS, Si@2DIS and Si@3DIS) immobilized on substrates and their palladacycle monolayers (Si@1DIS-Pd, Si@2DIS-Pd and Si@3DIS-Pd) were prepared and characterized. Their catalytic performances were investigated using the Suzuki coupling reaction as a model. Si@3DIS-Pd showed the highest catalytic activity in water without ligands, and better recyclability than that of Si@2DIS-Pd and Si@1DIS-Pd. The reason was the carbon in the aliphatic diimine of Si@2DIS-Pd and Si@1DIS-Pd was easily hydrolyzed because of the active hydrogen of α-C, resulting in poor recyclability. Control of the amount of catalyst could be achieved by modulating the diameter of the channel-like structure, which also affected the catalytic activity. The catalytic process and mechanism were investigated systematically and proposed based on the experimental results obtained by the water contact angle, ultraviolet spectroscopy, X-ray photoelectron spectroscopy, cyclic voltammetry and atomic force spectroscopy. Changes in the morphology of monolayer surfaces during the catalytic process with or without stirring presented a clear process from order to disorder, and indicated that the reaction was a heterogeneous catalytic process occurring on the surface of the catalyst monolayer.
| S-EPMC9085729 | biostudies-literature
Project description:Water oxidation is a linchpin in solar fuels formation, and catalysis by single-site ruthenium complexes has generated significant interest in this area. Combining several theoretical tools, we have studied the entire catalytic cycle of water oxidation for a single-site catalyst starting with [Ru(II)(tpy)(bpm)(OH(2))](2+) (i.e., [Ru(II)-OH(2)](2+); tpy is 2,2':6',2''-terpyridine and bpm is 2,2'-bypyrimidine) as a representative example of a new class of single-site catalysts. The redox potentials and pK(a) calculations for the first two proton-coupled electron transfers (PCETs) from [Ru(II)-OH(2)](2+) to [Ru(IV) = O](2+) and the following electron-transfer process to [Ru(V) = O](3+) suggest that these processes can proceed readily in acidic or weakly basic conditions. The subsequent water splitting process involves two water molecules, [Ru(V) = O](3+) to generate [Ru(III)-OOH](2+), and H(3)O(+) with a low activation barrier (~10 kcal/mol). After the key O-O bond forming step in the single-site Ru catalysis, another PECT process oxidizes [Ru(III)-OOH](2+) to [Ru(IV)-OO](2+) when the pH is lower than 3.7. Two possible forms of [Ru(IV)-OO](2+), open and closed, can exist and interconvert with a low activation barrier (< 7 kcal/mol) due to strong spin-orbital coupling effects. In Pathway 1 at pH = 1.0, oxygen release is rate-limiting with an activation barrier ~12 kcal/mol while the electron-transfer step from [Ru(IV)-OO](2+) to [Ru(V)-OO](3+) becomes rate-determining at pH = 0 (Pathway 2) with Ce(IV) as oxidant. The results of these theoretical studies with atomistic details have revealed subtle details of reaction mechanisms at several stages during the catalytic cycle. This understanding is helpful in the design of new catalysts for water oxidation.
| S-EPMC3465425 | biostudies-literature