The stereospecificity of alpha-chymotrypsin.
Ontology highlight
ABSTRACT:
SUBMITTER: Ingles DW
PROVIDER: S-EPMC1198851 | biostudies-other | 1968 Jul
REPOSITORIES: biostudies-other
Similar Datasets
Project description:1. The optically pure p-nitrophenyl esters of the d and l enantiomers of N-acetyl-tryptophan, N-acetylphenylalanine and N-acetyl-leucine, and the p-nitrophenyl ester of N-acetylglycine, have been prepared. 2. These materials are all substrates of alpha-chymotrypsin, and the rates of deacylation of the corresponding acyl-alpha-chymotrypsins have been determined. 3. As the size of the amino acid side chain increases, the l series deacylate progressively faster than the N-acetylglycyl-enzyme, and the d series progressively more slowly. 4. The results are interpreted in terms of a three-locus model of the enzyme's active site, which accounts for the interrelationship between substrate specificity and stereospecificity observed. 5. The concepts of negative specificity and of specificity saturation are introduced.
| S-EPMC1270596 | biostudies-other
Project description:Studies of salt effects on enzyme activity have typically been conducted at standard temperatures and pressures, thus missing effects which only become apparent under non-standard conditions. Here we show that perchlorate salts, which are found pervasively on Mars, increase the activity of α-chymotrypsin at low temperatures. The low temperature activation is facilitated by a reduced enthalpy of activation owing to the destabilising effects of perchlorate salts. By destabilising α-chymotrypsin, the perchlorate salts also cause an increasingly negative entropy of activation, which drives the reduction of enzyme activity at higher temperatures. We have also shown that α-chymotrypsin activity appears to exhibit an altered pressure response at low temperatures while also maintaining stability at high pressures and sub-zero temperatures. As the effects of perchlorate salts on the thermodynamics of α-chymotrypsin's activity closely resemble those of psychrophilic adaptations, it suggests that the presence of chaotropic molecules may be beneficial to life operating in low temperature environments.
| S-EPMC8367967 | biostudies-literature
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.
| S-EPMC7532203 | biostudies-literature
Project description:Precipitation of alpha chymotrypsin in the simultaneous presence of ammonium sulphate and t-butanol (three phase partitioning) resulted in preparations which showed self aggregation of the enzyme molecules. Precipitation with increasing amounts of ammonium sulphate led to increasing size of aggregates. While light scattering estimated the hydrodynamic diameter of these aggregates in the range of 242-1124 nm; Nanoparticle tracking analysis (NTA) gave the value as 130-462 nm. Scanning electron microscopy and gel filtration on Sephadex G-200 showed extensive aggregation in these preparations. Transmission electron microscopy showed that the aggregates had irregular shapes. All the aggregates had about 3× higher catalytic activity than the native enzyme. These aggregates did not differ in ?(max) of fluorescence emission which was around 340 nm. However, all the aggregates showed higher fluorescence emission intensity. Far-UV and near-UV circular dichroism also showed no significant structural changes as compared to the native molecule. Interestingly, HPLC gel filtration (on a hydroxylated silica column) gave 14 nm as the diameter for all preparations. Light scattering of preparations in the presence of 10% ethylene glycol also dissociated the aggregates to monomers of 14 nm. Both these results indicated that hydrophobic interactions were the driving force behind this aggregation. These results indicate: (1) Even without any major structural change, three phase partitioning led to protein molecules becoming highly prone to aggregation. (2) Different methods gave widely different estimates of sizes of aggregates. It was however possible to reconcile the data obtained with various approaches. (3) The nature of the gel filtration column is crucial and use of this technique for refolding and studying aggregation needs a rethink.
| S-EPMC3519768 | biostudies-literature
Project description:1. The binding of three competitive inhibitors, N-acetyl-d-tryptophan, N-acetyl-l-tryptophan and N-acetyl-d-tryptophan amide, to alpha-chymotrypsin was studied over the pH range 2.20-9.65 by the technique of equilibrium dialysis. 2. Within the limits of the experimental method, the binding of the uncharged amide inhibitor is independent of pH over the range investigated. 3. The binding of each of the enantiomeric acids is dependent on the ionization of a group on the free enzyme, of apparent pK(a)7.3. 4. It is shown that the ionizing group results in the active site of the enzyme developing a net negative charge above pH7.3. 5. The enzyme groups responsible are tentatively identified, and the significance of the binding constants with respect to the enzymic catalysis is discussed.
| S-EPMC1270065 | biostudies-other
Project description:BackgroundProtein instability remains the main factor limiting the development of protein therapeutics. The fragile nature (structurally and chemically) of proteins makes them susceptible to detrimental events during processing, storage, and delivery. To overcome this, proteins are often formulated in the solid-state which combines superior stability properties with reduced operational costs. Nevertheless, solid protein pharmaceuticals can also suffer from instability problems due to moisture sorption. Chemical protein glycosylation has evolved into an important tool to overcome several instability issues associated with proteins. Herein, we employed chemical glycosylation to stabilize a solid-state protein formulation against moisture-induced deterioration in the lyophilized state.ResultsFirst, we investigated the consequences of moisture sorption on the stability and structural conformation of the model enzyme alpha-chymotrypsin (alpha-CT) under controlled humidity conditions. Results showed that alpha-CT aggregates and inactivates as a function of increased relative humidity (RH). Furthermore, alpha-CT loses its native secondary and tertiary structure rapidly at increasing RH. In addition, H/D exchange studies revealed that alpha-CT structural dynamics increased at increasing RH. The magnitude of the structural changes in tendency parallels the solid-state instability data (i.e., formation of buffer-insoluble aggregates, inactivation, and loss of native conformation upon reconstitution). To determine if these moisture-induced instability issues could be ameliorated by chemical glycosylation we proceeded to modify our model protein with chemically activated glycans of differing lengths (lactose and dextran (10 kDa)). The various glycoconjugates showed a marked decrease in aggregation and an increase in residual activity after incubation. These stabilization effects were found to be independent of the glycan size.ConclusionWater sorption leads to aggregation, inactivation, and structural changes of alpha-CT as has been similarly shown to occur for many other proteins. These instabilities correlate with an increase in protein structural dynamics as a result of moisture exposure. In this work, we present a novel methodology to stabilize proteins against structural perturbations in the solid-state since chemical glycosylation was effective in decreasing and/or preventing the traditionally observed moisture-induced aggregation and inactivation. It is suggested that the stabilization provided by these chemically attached glycans comes from the steric hindrance that the sugars conveys on the protein surface therefore preventing the interaction of the protein internal electrostatics with that of the water molecules and thus reducing the protein structural dynamics upon moisture exposure.
| S-EPMC2924255 | biostudies-literature
Project description:Amyloid nanofibril formation appears to be a generic property of polypeptide chains. alpha-Chymotrypsin (aCT) was recently driven toward amyloid-like aggregation by the addition of trifluoroethanol (TFE) at intermediate concentrations. In this study we employed a molecular dynamics simulation to investigate the early events in TFE-induced conformational changes of aCT that precede amyloid formation, and compared the results of the simulation with previous experiments. TFE molecules were found to rapidly replace the water molecules closely associated with the protein surface. The gyration radius, together with total and hydrophobic solvent-accessible surface areas of aCT, was significantly increased. In accord with the experimental observations, the extended beta-conformation of backbone was increased. The secondary structural elements of aCT in water and TFE/water mixture showed a reasonable fit, whereas significant deviations were observed for several loops. These alterations originated largely from main-chain rotations at glycine residues. The catalytic active site and S1 binding pocket of the enzyme were also distorted in the TFE/water mixture. The obtained results are suggested to provide more insights into the conformational properties of the amyloid aggregation-prone protein species. Possible mechanisms of TFE-induced alterations in the conformation and dynamics of the protein structure are also discussed.
| S-EPMC2567952 | biostudies-literature
Project description:alpha-Chymotrypsin (EC 3.4.21.1)-catalysed syntheses of peptides were performed with various N-acylated amino acid or peptide esters as donors, and amino acid derivatives, peptides or their derivatives as acceptors. Under optimal conditions the synthesis was almost quantitative. As acceptor nucleophiles, free amino acids or the ester derivatives were inadequate, but amino acid amides or hydrazides, di- or tri-peptides, or the amides, hydrazides and esters of the peptides were useful. The nucleophile specificity for synthesis was markedly similar to the leaving-group specificity in hydrolysis; hydrophobic or bulky amino acid residues were most effecient at both P1' and P2' positions [notation of Schechter & Berger (1967) Biochem. Biophys. Res. Commun. 27, 157-162], but L-proline as well as D-amino acid residues were the worst choices. The synthesis was further dependent on the solubility of the products synthesized; a higher yield of products was expected with lower solubility. As donor esters, good substrates were all useful. Accordingly, fragment condensation was possible by using N-acylated peptide esters and various peptides. The present study suggested that alpha-chymotrypsin may become a useful tool for peptide synthesis.
| S-EPMC1164734 | biostudies-other
Project description:We recorded the far- and near-UV circular dichroism (CD) spectra of solutions of α-chymotrypsin and sodium dodecyl sulfate (SDS) with the final surfactant concentration significantly above the critical micellization concentration. Solutions were prepared using three different procedures. The reference procedure was to mix the chymotrypsin solution with the SDS solution once, immediately achieving the final SDS concentration. In alternative procedures, the protein solutions initially contained some SDS and were mixed with pure SDS solutions at a concentration to provide the same final surfactant as the reference mixing. We demonstrate that the supplementation to the selected final concentration of SDS of the pure chymotrypsin solution leads to different CD spectra than the supplementation to this final concentration of SDS in the chymotrypsin solution containing a small concentration of a few millimolar SDS. These differences disappear when the initial concentration of SDS in the protein solution, which we then supplement to the indicated final concentration, is higher. This suggests the irreversibility of the processes caused by the addition of SDS to chymotrypsin and the influence of the initial amount of this surfactant on the processes occurring with its further addition to the solution. For quantitative analysis of far-UV CD spectra in terms of populations of protein secondary structure elements, we used four well-established software packages. All programs consistently indicate that the observed differences in the far-UV CD spectra can be explained by the differences in the increase in the population of helical forms in chymotrypsin under the influence of SDS.
| S-EPMC9280762 | biostudies-literature
Project description:1. The reaction of alpha-chymotrypsin with sodium periodate at pH5.0 has been investigated. The enzyme consumes 2 moles of periodate/mole, and there is a concomitant fall in enzymic activity (with respect to l-tyrosine ethyl ester) to 55% of that of the native enzyme. After 3hr. no further change is observed in periodate uptake or in catalytic activity. 2. The oxidized enzyme is a homogeneous preparation of partially active chymotrypsin. 3. In the oxidized enzyme, one of the two methionine residues in the molecule has been converted into its sulphoxide. It is this reaction only that is responsible for the loss of activity. 4. The rate constants for the enzyme-catalysed acylation and deacylation reactions are unaltered by oxidation of the enzyme, both for a non-specific substrate (p-nitrophenyl acetate), and for three specific substrates: N-acetyl-l-tryptophan ethyl ester, N-acetyl-l-tryptophanamide and N-acetyl-l-valine ethyl ester. 5. The K(m) values for the aromatic substrates with the oxidized enzyme are twice those with the native enzyme. No change in Michaelis constant is seen for the non-aromatic substrate N-acetyl-l-valine ethyl ester. 6. The evidence points to the oxidized methionine residue in the modified enzyme being situated in the locus of the active site at which aromatic (or bulky) side chains of the substrates are bound.
| S-EPMC1215192 | biostudies-other