Pronase catalysed peptide syntheses

A mixture of proteases from Streptomyces griseus (pronase), displaying a very broad substrate tolerance in the hydrolysis of peptides, has been studied for the first time systematically regarding their substrate specificity in peptide synthesis, It is demonstrated that pronase can be employed successfully for the formation of dipeptides with yields up to 95%, Pronase has also been employed successfully as catalyst for the enzyme assisted synthesis of a hexapeptide

A conserved arginine plays a role in the catalytic cycle of the protein disulphide isomerases

The pKa values of the CXXC active-site cysteine residues play a critical role in determining the physiological function of the thioredoxin superfamily. To act as an efficient thiol-disulphide oxidant the thiolate state of the N-terminal cysteine must be stabilised and the thiolate state of the C-terminal cysteine residue destabilised. While increasing the pKa value of the C-terminal cysteine residue promotes oxidation of substrates, it has an inhibitory effect on the reoxidation of the enzyme, which is promoted by the formation of a thiolate at this position. Since reoxidation is essential to complete the catalytic cycle, the differential requirement for a high and a low pKa value for the C-terminal cysteine residue for different steps in the reaction presents us with a paradox. Here, we report the identification of a conserved arginine residue, located in the loop between β5 and α4 of the catalytic domains of the human protein disulphide isomerase (PDI) family, which is critical for the catalytic function of PDI, ERp57, ERp72 and P5, specifically for reoxidation. An examination of the published NMR structure for the a domain of PDI combined with molecular dynamic studies suggest that the side-chain of this arginine residue moves into and out of the active-site locale and that this has a very marked effect on the pKa value of the active-site cysteine residues. This intra-domain motion resolves the apparent dichotomy of the pKa requirements for the C-terminal active-site cysteine. © 2003 Elsevier Ltd. All rights reserved.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Best of Both Worlds: Combining Pharma Data and State of the Art Modeling Technology To Improve <i>in Silico</i> p<i>K</i>_a Prediction

In a unique collaboration between a software company and a pharmaceutical company, we were able to develop a new <i>in silico</i> p<i>K</i>_a prediction tool with outstanding prediction quality. An existing p<i>K</i>_a prediction method from Simulations Plus based on artificial neural network ensembles (ANNE), microstates analysis, and literature data was retrained with a large homogeneous data set of drug-like molecules from Bayer. The new model was thus built with curated sets of ∼14,000 literature p<i>K</i>_a values (∼11,000 compounds, representing literature chemical space) and ∼19,500 p<i>K</i>_a values experimentally determined at Bayer Pharma (∼16,000 compounds, representing industry chemical space). Model validation was performed with several test sets consisting of a total of ∼31,000 new p<i>K</i>_a values measured at Bayer. For the largest and most difficult test set with >16,000 p<i>K</i>_a values that were not used for training, the original model achieved a mean absolute error (MAE) of 0.72, root-mean-square error (RMSE) of 0.94, and squared correlation coefficient (<i>R</i>²) of 0.87. The new model achieves significantly improved prediction statistics, with MAE = 0.50, RMSE = 0.67, and <i>R</i>² = 0.93. It is commercially available as part of the Simulations Plus ADMET Predictor release 7.0. Good predictions are only of value when delivered effectively to those who can use them. The new p<i>K</i>_a prediction model has been integrated into Pipeline Pilot and the PharmacophorInformatics (PIx) platform used by scientists at Bayer Pharma. Different output formats allow customized application by medicinal chemists, physical chemists, and computational chemists

Molecular characterization of the principal substrate binding site of the ubiquitous folding catalyst protein disulfide isomerase.

Disulfide bond formation in the endoplasmic reticulum of eukaryotes is catalyzed by the ubiquitously expressed enzyme protein disulfide isomerase (PDI). The effectiveness of PDI as a catalyst of native disulfide bond formation in folding polypeptides depends on the ability to catalyze disulfide-dithiol exchange, to bind non-native proteins, and to trigger conformational changes in the bound substrate, allowing access to buried cysteine residues. It is known that the b' domain of PDI provides the principal peptide binding site of PDI and that this domain is critical for catalysis of isomerization but not oxidation reactions in protein substrates. Here we use homology modeling to define more precisely the boundaries of the b' domain and show the existence of an intradomain linker between the b' and a' domains. We have expressed the recombinant b' domain thus defined; the stability and conformational properties of the recombinant product confirm the validity of the domain boundaries. We have modeled the tertiary structure of the b' domain and identified the primary substrate binding site within it. Mutations within this site, expressed both in the isolated domain and in full-length PDI, greatly reduce the binding affinity for small peptide substrates, with the greatest effect being I272W, a mutation that appears to have no structural effect

Constructing regionalism in South America: the cases of sectoral cooperation on transport infrastructure and energy

First online: 29 January 2018This article contributes to the study of South American regionalism focusing on the emergence of sectoral cooperation starting in 2000. To do so, the article analyses two policy areas transport infrastructure and energy integration-addressing two questions: Why has regional cooperation emerged despite the absence of economic interdependence and market driven demand for economic integration? And why are policy outcomes evident in some areas (i.e. transport infrastructure) while limited in others (i.e. energy)? It is argued that the emergence of regional cooperation as well as the variation in policy outcomes between areas can be explained largely by the articulation of a regional leadership and its effect on the convergence of state preferences. The article shows how the Brazilian leadership, incentivised by the effects of the US-led Free Trade Area of the Americas negotiations and the financial crises that hit the region in the late 1990s, made state preferences converge towards a regionalist project encompassing all South American countries by making visible the mutual benefits of cooperation on transport infrastructure and energy. In the case of energy, however, the emergence of a second regional leadership project - pursued by Chavez's Venezuela- and deep preference divergence led sectoral cooperation into a gridlock