Allomorphy as a mechanism of post-translational control of enzyme activity

Enzyme regulation is vital for metabolic adaptability in living systems. Fine control of enzyme activity is often delivered through post-translational mechanisms, such as allostery or allokairy. β-phosphoglucomutase (βPGM) from Lactococcus lactis is a phosphoryl transfer enzyme required for complete catabolism of trehalose and maltose, through the isomerisation of β-glucose 1-phosphate to glucose 6-phosphate via β-glucose 1,6-bisphosphate. Surprisingly for a gatekeeper of glycolysis, no fine control mechanism of βPGM has yet been reported. Herein, we describe allomorphy, a post-translational control mechanism of enzyme activity. In βPGM, isomerisation of the K145-P146 peptide bond results in the population of two conformers that have different activities owing to repositioning of the K145 sidechain. In vivo phosphorylating agents, such as fructose 1,6-bisphosphate, generate phosphorylated forms of both conformers, leading to a lag phase in activity until the more active phosphorylated conformer dominates. In contrast, the reaction intermediate β-glucose 1,6-bisphosphate, whose concentration depends on the β-glucose 1-phosphate concentration, couples the conformational switch and the phosphorylation step, resulting in the rapid generation of the more active phosphorylated conformer. In enabling different behaviours for different allomorphic activators, allomorphy allows an organism to maximise its responsiveness to environmental changes while minimising the diversion of valuable metabolites

Evaluating the Impact of Nature-Based Solutions: A Handbook for Practitioners

The Handbook aims to provide decision-makers with a comprehensive NBS impact assessment framework, and a robust set of indicators and methodologies to assess impacts of nature-based solutions across 12 societal challenge areas: Climate Resilience; Water Management; Natural and Climate Hazards; Green Space Management; Biodiversity; Air Quality; Place Regeneration; Knowledge and Social Capacity Building for Sustainable Urban Transformation; Participatory Planning and Governance; Social Justice and Social Cohesion; Health and Well-being; New Economic Opportunities and Green Jobs. Indicators have been developed collaboratively by representatives of 17 individual EU-funded NBS projects and collaborating institutions such as the EEA and JRC, as part of the European Taskforce for NBS Impact Assessment, with the four-fold objective of: serving as a reference for relevant EU policies and activities; orient urban practitioners in developing robust impact evaluation frameworks for nature-based solutions at different scales; expand upon the pioneering work of the EKLIPSE framework by providing a comprehensive set of indicators and methodologies; and build the European evidence base regarding NBS impacts. They reflect the state of the art in current scientific research on impacts of nature-based solutions and valid and standardized methods of assessment, as well as the state of play in urban implementation of evaluation frameworks

Conviviality by design : the socio-spatial qualities of spaces of intercultural urban encounters

This paper presents findings from a mixed-method research project which explored use of outdoor spaces and social connections in Bradford, a post-industrial city in the north of England with a highly ethnically diverse population. Data was collected through micro-scale behavioural mapping of public spaces (analysed using GIS) and both on-site and in-depth interviews. The integration of these methods allows a focus on intersectional identities and social values for everyday conviviality situated in different typologies of public open spaces (parks, squares, streets) in city centre and suburban neighbourhoods. The analysis offers nuanced insights into the socio-spatial aspects of conviviality: patterns of activity by diverse users, situations in which encounters are prompted, and the implications of negotiating differences in relation to perceptions of self, others, and the environment. We discuss the relevance of the urban public realm for shared understandings of diversity, qualities of visibility, lingering and playfulness, and the importance of threshold spaces. We explore racialised and excluding experiences and how these relate to mobility and territorial patterns of use, specifically with relation to gender. The paper highlights connections between intercultural encounters and urban design practice, with implications for well-being and integration in ethnically diverse urban areas

Cholinesterases: Structure, Role, and Inhibition

Acetilkolinesteraza (AChE; E.C. 3.1.1.7) i butirilkolinesteraza (BChE; E.C. 3.1.1.8) enzimi su koji se zbog svoje uloge u organizmu intenzivno istražuju unutar područja biomedicine i toksikologije. Iako strukturno homologni, ovi enzimi razlikuju se prema katalitičkoj aktivnosti, odnosno specifi čnosti prema supstratima koje mogu hidrolizirati te selektivnosti za vezanje mnogih liganada. U ovom radu dan je pregled dosadašnjih istraživanja kolinesteraza i njihovih interakcija s ligandima i inhibitorima te su izdvojene aminokiseline aktivnog mjesta koje sudjeluju u tim interakcijama.Enzymes acetylcholinesterase (AChE; E.C. 3.1.1.7) and butyrylcholinesterase (BChE; E.C. 3.1.1.8) have intensively been investigated in biomedicine and toxicology due to important role in organisms. Even if structurally homologous, they differ in catalytic activity, specificity, for substrates, and selectivity in binding to many ligands. This paper compiles the results of research on cholinesterases and their interactions with ligands and inhibitors, and identifies amino acids of active sites involved in these interactions

Time-course of enzyme-catalyzed competing substrate degradation for michaelian behavior and for enzymes showing activation/inhibition by excess substrate

© 2019 Elsevier B.V. Progress curves for competing substrates were analyzed to investigate the effect of an “invisible” substrate (B) on the time-course of enzyme-catalyzed substrate degradation of a “visible” (reporter) substrate (A). Rate equations were integrated for Michaelis-Menten kinetics and in the case of activation or inhibition of degradation of A by excess of substrate The shape of progress curves depends on the ratio of specificity constants (kcat/Km)B/A, the competition matrix (R). Mathematical solutions exist for R ≫ 1, R = 1, R ≪ 1. Working at constant reporter substrate A concentration, from the shape of progress curves (sigmoidal or non-sigmoidal), it is possible to define the type of competitor (B), and from the dependence of retardation time (at 90% completion of A, and at inflexion point for sigmoid-like shaped progress curves) on “invisible” substrate B concentration, it is therefore possible to access to catalytic parameters, and/or to titrate enzyme active This competing substrate approach is suitable for investigating new substrates and reversible inhibitors of toxicological and pharmacological interest, investigating enzyme promiscuity, screening of enzymes degrading numerous compounds, and mining new enzymes of medical or biotechnological interest

Time-course of enzyme-catalyzed competing substrate degradation for michaelian behavior and for enzymes showing activation/inhibition by excess substrate

© 2019 Elsevier B.V. Progress curves for competing substrates were analyzed to investigate the effect of an “invisible” substrate (B) on the time-course of enzyme-catalyzed substrate degradation of a “visible” (reporter) substrate (A). Rate equations were integrated for Michaelis-Menten kinetics and in the case of activation or inhibition of degradation of A by excess of substrate The shape of progress curves depends on the ratio of specificity constants (kcat/Km)B/A, the competition matrix (R). Mathematical solutions exist for R ≫ 1, R = 1, R ≪ 1. Working at constant reporter substrate A concentration, from the shape of progress curves (sigmoidal or non-sigmoidal), it is possible to define the type of competitor (B), and from the dependence of retardation time (at 90% completion of A, and at inflexion point for sigmoid-like shaped progress curves) on “invisible” substrate B concentration, it is therefore possible to access to catalytic parameters, and/or to titrate enzyme active This competing substrate approach is suitable for investigating new substrates and reversible inhibitors of toxicological and pharmacological interest, investigating enzyme promiscuity, screening of enzymes degrading numerous compounds, and mining new enzymes of medical or biotechnological interest

Time-course of human cholinesterases-catalyzed competing substrate kinetics

© 2019 Elsevier B.V. Competing substrate kinetic analysis of human butyrylcholinesterase (BChE) and acetylcholinesterase (AChE) from the time-course of enzyme-catalyzed substrate hydrolysis, using spectrophotometric assays is described. This study is based on the use of a chromogenic reporter “visible” substrate (substrate A), whose complete hydrolysis time course is retarded by a competing “invisible” substrate (substrate B). For BChE, four visible substrates were used, two thiocholine esters, benzoylthiocholine and butyrylthiocholine, and two aryl-acylamides, o-nitro trifluoro acetaminide and 3-(acetamido)-N,N,N-trimethylanilinium. Three different competing invisible substrates were used, phenyl acetate, acetylcholine and butyrylcholine. For AChE, two visible substrates were used, acetylthiocholine and 3-(acetamido)-N,N,N-trimethylanilinium. For AChE, acetylcholine was competing with visible substrates. The ratio (R) of bimolecular rate constants, kcat/Km, for all couples of substrates, invisible/visible (B/A) covered all possible limit situations, R ≪ 1, R ≈ 1 and R ≫ 1. The kinetic approach, based on the method developed by Golicnik and Masson allowed determination of binding and catalytic parameters of cholinesterases for both visible and invisible substrates. This analysis was applied to michaelian and non-michaelian catalytic behaviors (activation and inhibition by excess substrate). Reevaluation of catalytic parameters obtained for acetylcholine and butyrylcholine more than 50 years ago was made. The method is fast, reliable, and particularly suitable for poorly soluble substrates and for substrates B when no direct spectrophotometric assays exist. Moreover, replacing substrate B by a reversible inhibitor, mechanism of cholinesterase inhibition was possible to study. It is therefore, useful for screening libraries of new substrates and inhibitors, and/or screening of new cholinesterase mutants. This method can be applied to any other enzymes

Time-course of human cholinesterases-catalyzed competing substrate kinetics

© 2019 Elsevier B.V. Competing substrate kinetic analysis of human butyrylcholinesterase (BChE) and acetylcholinesterase (AChE) from the time-course of enzyme-catalyzed substrate hydrolysis, using spectrophotometric assays is described. This study is based on the use of a chromogenic reporter “visible” substrate (substrate A), whose complete hydrolysis time course is retarded by a competing “invisible” substrate (substrate B). For BChE, four visible substrates were used, two thiocholine esters, benzoylthiocholine and butyrylthiocholine, and two aryl-acylamides, o-nitro trifluoro acetaminide and 3-(acetamido)-N,N,N-trimethylanilinium. Three different competing invisible substrates were used, phenyl acetate, acetylcholine and butyrylcholine. For AChE, two visible substrates were used, acetylthiocholine and 3-(acetamido)-N,N,N-trimethylanilinium. For AChE, acetylcholine was competing with visible substrates. The ratio (R) of bimolecular rate constants, kcat/Km, for all couples of substrates, invisible/visible (B/A) covered all possible limit situations, R ≪ 1, R ≈ 1 and R ≫ 1. The kinetic approach, based on the method developed by Golicnik and Masson allowed determination of binding and catalytic parameters of cholinesterases for both visible and invisible substrates. This analysis was applied to michaelian and non-michaelian catalytic behaviors (activation and inhibition by excess substrate). Reevaluation of catalytic parameters obtained for acetylcholine and butyrylcholine more than 50 years ago was made. The method is fast, reliable, and particularly suitable for poorly soluble substrates and for substrates B when no direct spectrophotometric assays exist. Moreover, replacing substrate B by a reversible inhibitor, mechanism of cholinesterase inhibition was possible to study. It is therefore, useful for screening libraries of new substrates and inhibitors, and/or screening of new cholinesterase mutants. This method can be applied to any other enzymes