Catalytic activity of -chymotrypsin in which histidine-57 has been methylated.
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ABSTRACT:
SUBMITTER: Henderson R
PROVIDER: S-EPMC1177107 | biostudies-other | 1971 Aug
REPOSITORIES: biostudies-other
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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.
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Project description:The use of a linear free-energy relationship shows that both histidine residues of alpha-chymotrypsin and chymotrypsinogen are super-reactive toward 1-fluoro-2,4-dinitrobenzene. The binding of indole to the specificity site of alpha-chymotrypsin causes both histidine residues to become less reactive. On the basis of these results and those from X-ray-crystallographic studies, the following conclusions are made. (1) The super-reactivity of the catalytic-site histidine-57 is due to charge transfer from aspartic acid-102 by means of hydrogen bonding. (2) The aspartic acid-102-histidine-57-serine-195 'charge-relay' system is not complete in the zymogen or native enzyme and only on binding of a suitable substrate or ligand to the specificity site of the enzyme is the charge transfer to serine-195 completed. (3) The lack of substantial enzymic activity in the zymogen is due to the absence of a completed specificity site, and therefore it cannot bind suitable substrates or ligands to induce completion of the charge-relay system.
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Project description:The lethal factor (LF) of Bacillus anthracis is a Zn(2+)-dependent metalloprotease which plays an important role in anthrax virulence. This study was aimed at identifying the histidine residues that are essential to the catalytic activities of LF. The site-directed mutagenesis was employed to replace the 10 histidine residues in domains II, III, and IV of LF with alanine residues, respectively. The cytotoxicity of these mutants was tested, and the results revealed that the alanine substitution for His-669 completely abolished toxicity to the lethal toxin (LT)-sensitive RAW264.7 cells. The reason for the toxicity loss was further explored. The zinc content of this LF mutant was the same as that of the wild type. Also this LF mutant retained its protective antigan (PA)-binding activity. Finally, the catalytic cleavage activity of this mutant was demonstrated to be drastically reduced. Thus, we conclude that residue His-669 is crucial to the proteolytic activity of LF.
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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.
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Project description:Deep subsurface environments can harbour high concentrations of dissolved ions, yet we know little about how this shapes the conditions for life. We know even less about how the combined effects of high pressure influence the way in which ions constrain the possibilities for life. One such ion is perchlorate, which is found in extreme environments on Earth and pervasively on Mars. We investigated the interactions of high pressure and high perchlorate concentrations on enzymatic activity. We demonstrate that high pressures increase α-chymotrypsin enzyme activity even in the presence of high perchlorate concentrations. Perchlorate salts were shown to shift the folded α-chymotrypsin phase space to lower temperatures and pressures. The results presented here may suggest that high pressures increase the habitability of environments under perchlorate stress. Therefore, deep subsurface environments that combine these stressors, potentially including the subsurface of Mars, may be more habitable than previously thought.
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Project description:Substituting the P1 position in bovine pancreatic trypsin inhibitor (BPTI) is known to heavily influence its inhibitory activity towards serine proteases. Side-chain fluorinated aliphatic amino acids have been shown to alter numerous properties of peptides and proteins and thus are of interest in the context of BPTI. In our study, we systematically investigated the site-specific incorporation of non-canonical amino acids into BPTI by microwave-assisted solid-phase peptide synthesis (SPPS). Inhibitor activity of the variants was tested towards the serine protease α-chymotrypsin. We observed enhanced inhibition of two fluorinated BPTIs compared to wild type and hydrocarbon variants. To further investigate the complexes, we performed X-ray structure analysis. Our findings underline the power fluorine offers as a tool in protein engineering to beneficially alter the effects on phenomena as protein-protein interactions.
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Project description:Aldo-keto reductases (AKRs) are a major superfamily of monomeric NADPH-dependent carbonyl oxidoreductases. They are characterized by an (alpha/beta)(8)-barrel structure, which at its base contains a conserved catalytic tetrad of Tyr, Lys, His and Asp. Two AKR subfamilies contain other residues substituted for the catalytic His and perform different functions. First, the steroid 5beta-reductase (AKR1D1), which reduces CC double bonds instead of carbonyl groups, has a Glu substituted for His. Second, the Kvbeta subunits (AKR6A3, AKR6A5 and AKR6A9) which modulate opening of the voltage-gated potassium channel (Kv1) by oxidizing NADPH, have an Asn substituted for the His. Previously, we noted that conserved catalytic residues in AKRs perform similar functions in the short-chain dehydrogenases (SDRs). With the availability of crystal structures of AKR1D1 and two SDRs that catalyze double-bond reduction reactions, Digitalis steroid 5beta-reductase and 2,4-dienoyl-CoA reductase, we have compared their active sites to outline the features that govern whether 1,2-, 1,4- or 1,6-hydride transfer occurs.
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Project description:Histidine triad nucleotide binding proteins (Hints) are highly conserved members of the histidine triad (HIT) protein superfamily. Hints comprise the most ancient branch of this superfamily and can be found in Archaea, Bacteria, and Eukaryota. Prokaryotic genomes, including a wide diversity of both gram-negative and gram-positive bacteria, typically have one Hint gene encoded by hinT (ycfF in E. coli). Despite their ubiquity, the foundational reason for the wide-spread conservation of Hints across all kingdoms of life remains a mystery. In this study, we used a combination of phenotypic screening and complementation analyses with wild-type and hinT knock-out Escherichia coli strains to show that catalytically active ecHinT is required in E. coli for growth on D-alanine as a sole carbon source. We demonstrate that the expression of catalytically active ecHinT is essential for the activity of the enzyme D-alanine dehydrogenase (DadA) (equivalent to D-amino acid oxidase in eukaryotes), a necessary component of the D-alanine catabolic pathway. Site-directed mutagenesis studies revealed that catalytically active C-terminal mutants of ecHinT are unable to activate DadA activity. In addition, we have designed and synthesized the first cell-permeable inhibitor of ecHinT and demonstrated that the wild-type E. coli treated with the inhibitor exhibited the same phenotype observed for the hinT knock-out strain. These results reveal that the catalytic activity and structure of ecHinT is essential for DadA function and therefore alanine metabolism in E. coli. Moreover, they provide the first biochemical evidence linking the catalytic activity of this ubiquitous protein to the biological function of Hints in Escherichia coli.
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