On the water-promoted mechanism of peptide cleavage by carboxypeptidase A. A theoretical study

1994 ◽  
Vol 72 (10) ◽  
pp. 2077-2083 ◽  
Author(s):  
Silvia Alvarez-Santos ◽  
Angels González-Lafont ◽  
José M. Lluch ◽  
Baldomero Oliva ◽  
Francesc X. Avilés
Keyword(s):  
Theoretical Calculations ◽  
Peptide Bond ◽  
Proton Acceptor ◽  
Carboxypeptidase A ◽  
Peptide Cleavage ◽  
High Enthalpy ◽  
Rate Determining Step ◽  
Catalytic Mechanisms ◽  
Catalyzed Reactions ◽  
Enzyme Catalyzed Reactions

The water-promoted pathway of peptide cleavage by carboxypeptidase A has been studied by semiempirical (AM1) quantum mechanical calculations. A relatively large model for the CPA-active site elements plus substrate has been designed, using two imidazoles and one acetate as the Zn2+ ligands, acetate as the proton acceptor (simulating Glu-270), and N-ethylacetamide as the peptide-like substrate. This model, although simpler than the natural one, is one of the largest used for theoretical calculations on CPA catalytic mechanisms. To ensure that this model is able to mimic the natural system, it has been compared with the structure of the (Gly)3-L-Tyr + water + CPA complex resulting from several molecular dynamics/energy minimization simulations. Among the different steps involved in the water-promoted pathway proposed by Lipscomb's group, the attack of the oxygen atom that comes from the activated water molecule to the carbon atom of the peptide bond of the substrate has been found to be the rate-determining step, with a high enthalpy barrier of 37.9 kcal/mol. However, this enthalpy barrier is dramatically decreased when a positive charge, simulating Arg-127, is included near the scissile carbonyl. The reported results seem to favour the occurrence of the mechanism studied and indicate the limitations of using simple elements for the theoretical analysis of enzyme-catalyzed reactions.

scholarly journals Use of isotopically labeled substrates reveals kinetic differences between human and bacterial serine palmitoyltransferase

2019 ◽  
Vol 60 (5) ◽  
pp. 953-962 ◽  
Author(s):  
Peter J. Harrison ◽  
Kenneth Gable ◽  
Niranjanakumari Somashekarappa ◽  
Van Kelly ◽  
David J. Clarke ◽  
...  
Keyword(s):  
In Vivo Studies ◽  
Serine Palmitoyltransferase ◽  
Biochemical Pathways ◽  
Catalytic Mechanisms ◽  
Catalyzed Reactions ◽  
Enzyme Catalyzed Reactions ◽  
The Impact ◽  
Sphingolipid Biosynthesis

Isotope labels are frequently used tools to track metabolites through complex biochemical pathways and to discern the mechanisms of enzyme-catalyzed reactions. Isotopically labeled l-serine is often used to monitor the activity of the first enzyme in sphingolipid biosynthesis, serine palmitoyltransferase (SPT), as well as labeling downstream cellular metabolites. Intrigued by the effect that isotope labels may be having on SPT catalysis, we characterized the impact of different l-serine isotopologues on the catalytic activity of recombinant SPT isozymes from humans and the bacterium Sphingomonas paucimobilis. Our data show that S. paucimobilis SPT activity displays a clear isotope effect with [2,3,3-D]l-serine, whereas the human SPT isoform does not. This suggests that although both human and S. paucimobilis SPT catalyze the same chemical reaction, there may well be underlying subtle differences in their catalytic mechanisms. Our results suggest that it is the activating small subunits of human SPT that play a key role in these mechanistic variations. This study also highlights that it is important to consider the type and location of isotope labels on a substrate when they are to be used in in vitro and in vivo studies.

Theoretical Studies of Catalysis by Carboxypeptidase A: Could Gas-Phase Calculations Support a Mechanism?

2003 ◽  
Vol 68 (11) ◽  
pp. 2055-2079 ◽  
Author(s):  
Alexandra Kilshtain-Vardi ◽  
Gil Shoham ◽  
Amiram Goldblum
Keyword(s):  
Gas Phase ◽  
Peptide Bond ◽  
Main Question ◽  
Carboxypeptidase A ◽  
Peptide Cleavage ◽  
Alternative Pathways ◽  
General Base ◽  
General Acid ◽  
Reduced Representations ◽  
Quantum Mechanical Computations

We compare recent quantum mechanical computations of alternative reaction pathways for carboxypeptidase A, a zinc proteinase, in an "enzyme environment" to similar calculations in the "gas phase" that include the minimal chemical entities that are required for a non-catalytic reaction. The main question that we address is whether anything may be learned from such reduced representations. Two general acid-general base alternative pathways and one nucleophilic pathway are compared. The original calculations were run on a relatively large model (120 atoms) of the active site of carboxypeptidase A which included zinc and its ligands, as well as the residues Arg145, Arg127, Glu270, a water molecule and a model dipeptide. The "gas-phase" pathways include only the dipeptide, water and Glu270 and serve as models for the non-catalytic pathway. The calculations were performed by semiempirical MNDO/H/d that includes modifications for d-orbital representations as well as for intra- and intermolecular multiple H-bond formation. The gas-phase results strengthen our previous conclusion about the preference for general acid-general base pathways for peptide cleavage by carboxypeptidase A rather than a "direct nucleophilic" pathway. The bottleneck of the reaction is proton transfer to the nitrogen in the peptide bond, preceding the peptide cleavage.

A kinetic analysis of coupled sequential enzyme reactions

2018 ◽  
Author(s):  
Justin Eilertsen ◽  
Santiago Schnell
Keyword(s):  
Enzyme Assay ◽  
Sequential Reaction ◽  
Enzyme Reactions ◽  
Indicator Reaction ◽  
Single Substrate ◽  
Complex Sequences ◽  
Catalyzed Reactions ◽  
Enzyme Catalyzed Reactions ◽  
Single Enzyme

<div>As a case study, we consider a coupled enzyme assay of sequential enzyme reactions obeying the Michaelis--Menten reaction mechanism. The sequential reaction consists of a single-substrate, single-enzyme non-observable reaction followed by another single-substrate, single-enzyme observable reaction (indicator reaction). In this assay, the product of the non-observable reaction becomes the substrate of the indicator reaction. A mathematical analysis of the reaction kinetics is performed, and it is found that after an initial fast transient, the sequential reaction is described by a pair of interacting Michaelis--Menten equations. Timescales that approximate the respective lengths of the indicator and non-observable reactions, as well as conditions for the validity of the Michaelis--Menten equations are derived. The theory can be extended to deal with more complex sequences of enzyme catalyzed reactions.</div>

A kinetic analysis of coupled sequential enzyme reactions

2018 ◽  
Author(s):  
Justin Eilertsen ◽  
Santiago Schnell
Keyword(s):  
Enzyme Assay ◽  
Sequential Reaction ◽  
Enzyme Reactions ◽  
Indicator Reaction ◽  
Single Substrate ◽  
Complex Sequences ◽  
Catalyzed Reactions ◽  
Enzyme Catalyzed Reactions ◽  
Single Enzyme

<div>As a case study, we consider a coupled enzyme assay of sequential enzyme reactions obeying the Michaelis-Menten reaction mechanism. The sequential reaction consists of a single-substrate, single enzyme non-observable reaction followed by another single-substrate, single enzyme observable reaction (indicator reaction). In this assay, the product of the non-observable reaction becomes the substrate of the indicator reaction. A mathematical analysis of the reaction kinetics is performed, and it is found that after an initial fast transient, the sequential reaction is described by a pair of interacting Michaelis-Menten equations. Timescales that approximate the respective lengths of the indicator and non-observable reactions, as well as conditions for the validity of the Michaelis-Menten equations are derived. The theory can be extended to deal with more complex sequences of enzyme catalyzed reactions.</div>

Computationally Augmented Retrosynthesis: Total Synthesis of Paspaline A and Emindole PB

2018 ◽  
Author(s):  
Timothy Newhouse ◽  
Daria E. Kim ◽  
Joshua E. Zweig
Keyword(s):  
Chemical Synthesis ◽  
Total Synthesis ◽  
Small Molecules ◽  
First Principles ◽  
Quantum Chemical ◽  
Computational Analysis ◽  
Empirical Evaluation ◽  
Synthetic Strategy ◽  
Catalyzed Reactions ◽  
Enzyme Catalyzed Reactions

The diverse molecular architectures of terpene natural products are assembled by exquisite enzyme-catalyzed reactions. Successful recapitulation of these transformations using chemical synthesis is hard to predict from first principles and therefore challenging to execute. A means of evaluating the feasibility of such chemical reactions would greatly enable the development of concise syntheses of complex small molecules. Herein, we report the computational analysis of the energetic favorability of a key bio-inspired transformation, which we use to inform our synthetic strategy. This approach was applied to synthesize two constituents of the historically challenging indole diterpenoid class, resulting in a concise route to (–)-paspaline A in 9 steps from commercially available materials and the first pathway to and structural confirmation of emindole PB in 13 steps. This work highlights how traditional retrosynthetic design can be augmented with quantum chemical calculations to reveal energetically feasible synthetic disconnections, minimizing time-consuming and expensive empirical evaluation.

scholarly journals Diverse Taxonomies for Diverse Chemistries: Enhanced Representation of Natural Product Metabolism in UniProtKB

Metabolites ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 48
Author(s):  
Marc Feuermann ◽  
Emmanuel Boutet ◽  
Anne Morgat ◽  
Kristian Axelsen ◽  
Parit Bansal ◽  
...  
Keyword(s):  
Natural Products ◽  
Natural Product ◽  
Semantic Web Technologies ◽  
Knowledge Resources ◽  
Web Technologies ◽  
Chemical Structures ◽  
Biological Interest ◽  
Catalyzed Reactions ◽  
Enzyme Catalyzed Reactions ◽  
Practical Demonstration

The UniProt Knowledgebase UniProtKB is a comprehensive, high-quality, and freely accessible resource of protein sequences and functional annotation that covers genomes and proteomes from tens of thousands of taxa, including a broad range of plants and microorganisms producing natural products of medical, nutritional, and agronomical interest. Here we describe work that enhances the utility of UniProtKB as a support for both the study of natural products and for their discovery. The foundation of this work is an improved representation of natural product metabolism in UniProtKB using Rhea, an expert-curated knowledgebase of biochemical reactions, that is built on the ChEBI (Chemical Entities of Biological Interest) ontology of small molecules. Knowledge of natural products and precursors is captured in ChEBI, enzyme-catalyzed reactions in Rhea, and enzymes in UniProtKB/Swiss-Prot, thereby linking chemical structure data directly to protein knowledge. We provide a practical demonstration of how users can search UniProtKB for protein knowledge relevant to natural products through interactive or programmatic queries using metabolite names and synonyms, chemical identifiers, chemical classes, and chemical structures and show how to federate UniProtKB with other data and knowledge resources and tools using semantic web technologies such as RDF and SPARQL. All UniProtKB data are freely available for download in a broad range of formats for users to further mine or exploit as an annotation source, to enrich other natural product datasets and databases.

ChemInform Abstract: A Practical Guide to Modelling Enzyme-Catalyzed Reactions

ChemInform ◽  
2012 ◽  
Vol 43 (29) ◽  
pp. no-no
Author(s):  
Richard Lonsdale ◽  
Jeremy N. Harvey ◽  
Adrian J. Mulholland
Keyword(s):  
Practical Guide ◽  
Catalyzed Reactions ◽  
Enzyme Catalyzed Reactions
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