scholarly journals On Catalysis by Biological Macromolecular Enzymes

2020 ◽  
Author(s):  
Jianshu Dong
Keyword(s):  
Enzyme Kinetics ◽  
Enzyme Catalysis ◽  
Kinetic Data ◽  
Time Course ◽  
Molecular Level ◽  
Catalytic Efficiency ◽  
Molecular Behavior ◽  
Catalytic Rate

Classical enzyme kinetics are interpreted from a new angle here, and biological macromolecular enzyme catalysis is viewed and explored at the molecular level. The time course of sequential catalytic events is analyzed, the relationships between catalytic efficiency, catalytic rate/velocity and the amount of time consumed are established. This writing tries to connect the microscopic molecular behavior of enzymes to kinetic data obtained in experiment, and the equations proposed here can be testified and examined by future experiments.

scholarly journals On Catalysis by Biological Macromolecular Enzymes

2020 ◽  
Author(s):  
Jianshu Dong
Keyword(s):  
Enzyme Kinetics ◽  
Kinetic Data ◽  
Time Course ◽  
Molecular Level ◽  
Catalytic Efficiency ◽  
Turnover Number ◽  
Molecular Behavior ◽  
Catalytic Rate

Classical enzyme kinetics are summarized and linked with modern discoveries here. The time course of sequential catalytic events by biological macromolecular enzyme is analyzed at the molecular level; the relationships between catalytic efficiency (turnover number), catalytic rate/velocity, the amount of time taken and physical/biochemical conditions of the system are discussed. This writing tries to connect the microscopic molecular behavior of enzyme to kinetic data obtained in experiment, and the hypothesis proposed here provide an interpretation to previous experimental observations and can be testified by future experiments.

scholarly journals On Catalytic Kinetics of Enzymes

Processes ◽  
2021 ◽  
Vol 9 (2) ◽  
pp. 271
Author(s):  
Jianshu Dong
Keyword(s):  
Kinetic Data ◽  
Molecular Level ◽  
Chemical Conversion ◽  
Time Axis ◽  
Product Release ◽  
Molecular Behavior ◽  
Catalytic Kinetics ◽  
Catalytic Rate ◽  
The Times ◽  
Kinetics Of

Classical enzyme kinetic theories are summarized and linked with modern discoveries here. The sequential catalytic events along time axis by enzyme are analyzed at the molecular level, and by using master equations, this writing tries to connect the microscopic molecular behavior of enzyme to kinetic data (like velocity and catalytic coefficient k) obtained in experiment: 1/k = t equals to the sum of the times taken by the constituent individual steps. The relationships between catalytic coefficient k, catalytic rate or velocity, the amount of time taken by each step and physical or biochemical conditions of the system are discussed, and the perspective and hypothetic equations proposed here regarding diffusion, conformational change, chemical conversion, product release steps and the whole catalytic cycle provide an interpretation of previous experimental observations and can be testified by future experiments.

scholarly journals Analyzing Enzyme Kinetic Data Using the Powerful Statistical Capabilities of R

2018 ◽  
Author(s):  
Carly Huitema ◽  
Geoff Horsman
Keyword(s):  
Statistical Analysis ◽  
Enzyme Kinetics ◽  
Enzyme Catalysis ◽  
Kinetic Data ◽  
Goodness Of Fit ◽  
Linear Models ◽  
Command Line ◽  
Enzyme Kinetic ◽  
Non Linear ◽  
Statistical Program

AbstractWe describe a powerful tool for enzymologists to use for typical non-linear fitting of common equations in enzyme kinetics using the statistical program R. Enzyme kinetics is a powerful tool for understanding enzyme catalysis, regulation, and inhibition but tools to perform the analysis have limitations. Software to perform the necessary nonlinear analysis may be proprietary, expensive or difficult to use, especially for a beginner. The statistical program R is ideally suited to analyzing enzyme kinetic data; it is free in two respects: there is no cost and there is freedom to distribute and modify. It is also robust, powerful and widely used in other fields of biology. In this paper we introduce the program R to enzymologists who want to analyze their data but are unfamiliar with R or similar command line statistical analysis programs. Data are inputted and examples of different non-linear models are fitted. Results are extracted and plots are generated to assist judging the goodness of fit. The instructions will allow users to create their own modifications to adapt the protocol to their own experiments. Because of the use of scripts, a method can be modified and used to analyze different datasets in less than one hour.

scholarly journals AuNPs-Based Thermoresponsive Nanoreactor as an Efficient Catalyst for the Reduction of 4-Nitrophenol

Nanomaterials ◽  
2018 ◽  
Vol 8 (12) ◽  
pp. 963
Author(s):  
Wei Liu ◽  
Xiaolian Zhu ◽  
Chengcheng Xu ◽  
Zhao Dai ◽  
Zhaohui Meng
Keyword(s):  
Aqueous Solution ◽  
Electron Transfer ◽  
Phase Transformation ◽  
Catalytic Activity ◽  
Catalytic Efficiency ◽  
Environmental Pollutant ◽  
Temperature Sensitive ◽  
Efficient Catalyst ◽  
Catalytic Rate ◽  
Silica Core

A new AuNPs-based thermosensitive nanoreactor (SiO2@PMBA@Au@PNIPAM) was designed and prepared by stabilizing AuNPs in the layer of poly(N,N’-methylenebisacrylamide) (PMBA) and subsequent wrapping with the temperature-sensitive poly(N-isopropylacrylamide) (PNIPAM) layer. The new nanoreactor exhibited high dispersibility and stability in aqueous solution and effectively prevented the aggregation of AuNPs caused by the phase transformation of PNIPAM. The XPS and ATR-FTIR results indicated that AuNPs could be well stabilized by PMBA due to the electron transfer between the N atoms of amide groups in the PMBA and Au atoms of AuNPs. The catalytic activity and thermoresponsive property of the new nanoreactor were invested by the reduction of the environmental pollutant, 4-nitrophenol (4-NP), with NaBH4 as a reductant. It exhibited a higher catalytic activity at 20 °C and 30 °C (below LCST of PNIPAM), but an inhibited catalytic activity at 40 °C (above LCST of PNIPAM). The PNIPAM layer played a switching role in controlling the catalytic rate by altering the reaction temperature. In addition, this nanoreactor showed an easily recyclable property due to the existence of a silica core and also preserved a rather high catalytic efficiency after 16 times of recycling.

A Novel High-Throughput FLIPR Tetra–Based Method for Capturing Highly Confluent Kinetic Data for Structure–Kinetic Relationship Guided Early Drug Discovery

2021 ◽  
pp. 247255522110006
Author(s):  
Puneet Khurana ◽  
Lisa McWilliams ◽  
Jonathan Wingfield ◽  
Derek Barratt ◽  
Bharath Srinivasan
Keyword(s):  
Parameter Estimation ◽  
Drug Discovery ◽  
High Throughput ◽  
Kinetic Data ◽  
Time Course ◽  
Kinetic Studies ◽  
Discovery Process ◽  
Drug Discovery Process ◽  
Traditional Methods ◽  
Kinetic Relationship

Target engagement by small molecules is necessary for producing a physiological outcome. In the past, a lot of emphasis was placed on understanding the thermodynamics of such interactions to guide structure–activity relationships. It is becoming clearer, however, that understanding the kinetics of the interaction between a small-molecule inhibitor and the biological target [structure–kinetic relationship (SKR)] is critical for selection of the optimum candidate drug molecule for clinical trial. However, the acquisition of kinetic data in a high-throughput manner using traditional methods can be labor intensive, limiting the number of molecules that can be tested. As a result, in-depth kinetic studies are often carried out on only a small number of compounds, and usually at a later stage in the drug discovery process. Fundamentally, kinetic data should be used to drive key decisions much earlier in the drug discovery process, but the throughput limitations of traditional methods preclude this. A major limitation that hampers acquisition of high-throughput kinetic data is the technical challenge in collecting substantially confluent data points for accurate parameter estimation from time course analysis. Here, we describe the use of the fluorescent imaging plate reader (FLIPR), a charge-coupled device (CCD) camera technology, as a potential high-throughput tool for generating biochemical kinetic data with smaller time intervals. Subsequent to the design and optimization of the assay, we demonstrate the collection of highly confluent time-course data for various kinase protein targets with reasonable throughput to enable SKR-guided medicinal chemistry. We select kinase target 1 as a special case study with covalent inhibition, and demonstrate methods for rapid and detailed analysis of the resultant kinetic data for parameter estimation. In conclusion, this approach has the potential to enable rapid kinetic studies to be carried out on hundreds of compounds per week and drive project decisions with kinetic data at an early stage in drug discovery.

The study of enzyme mechanisms by a combination of cosolvent, low-temperature and high-pressure techniques

1979 ◽  
Vol 12 (4) ◽  
pp. 521-569 ◽  
Author(s):  
Pierre Douzou
Keyword(s):  
High Pressure ◽  
Conformational Changes ◽  
Rate Constants ◽  
Enzyme Catalysis ◽  
Chemical Nature ◽  
Molecular Level ◽  
Essential Feature ◽  
Quantitative Understanding ◽  
Definition Of ◽  
Rapid Data Acquisition

It is generally assumed that the mechanism of enzyme-catalysed reactions would be defined if all the intermediates, complexes and conformational states of each enzyme could be characterized, and the rate-constants for their inter-conversion recorded. In spite of the introduction during the last decades of methods for rapid data acquisition, which permit detection of the number and sequence of intermediates and complexes, measurement of rate-constants, identification of the types of catalysis involved, etc., at best a semi-quantitative understanding of the mechanism of enzyme-catalysis is obtained. The definition of the exact chemical nature of intermediates and complexes is missing because techniques establishing the structures are restricted to the study of transient states which are stable for periods that exceed the half-life of most of typical intermediates. In such conditions, while conformational changes are obviously an essential feature of enzyme activity, the conformational basis of such activity cannot be understood at the molecular level, and enzyme catalysis is still termed a ‘miracle’ compared to the rate-enhancements and specificity of ordinary chemical catalysts.

scholarly journals High catalytic rate of the cold-active Vibrio alkaline phosphatase depends on a hydrogen bonding network involving a large interface loop

2020 ◽  
Author(s):  
Jens Guðmundur Hjörleifsson ◽  
Ronny Helland ◽  
Manuela Magnúsdóttir ◽  
Bjarni Ásgeirsson
Keyword(s):  
Alkaline Phosphatase ◽  
Temperature Stability ◽  
Catalytic Efficiency ◽  
Phosphoryl Group ◽  
Vibrio Splendidus ◽  
Large Loop ◽  
Dynamic Mobility ◽  
Catalytic Rate ◽  
Surface Loops

AbstractThe role of surface loops in mediating communication through residue networks is still a relatively poorly understood part of cold-adaptation of enzymes, especially in terms of their quaternary interactions. Alkaline phosphatase (AP) from the psychrophilic marine bacterium Vibrio splendidus (VAP) is characterized by an analogous large surface loop in each monomer, referred to as the large-loop, that hovers over the active site of the other monomer. It presumably has a role in VAP high catalytic efficiency that accompanies extremely low thermal stability. We designed several different mutagenic variants of VAP with the aim of removing inter-subunit interactions at the dimer interface. Breaking the inter-subunit contacts from one residue in particular (Arg336) caused diminished temperature stability of the catalytically potent conformation and a drop in catalytic rate by a half. The relative B-factors of the R336L crystal structure, compared to the wild-type, confirmed increased surface flexibility in a loop on the opposite monomer, but not in the large-loop. Contrary to expectations, the observed reduction in stability with an expected increase in dynamic mobility resulted in reduced catalytic rate. This contradicts common theories explaining high catalytic rates of enzyme from cold-adapted organisms as being due to reduced internal cohesion bringing increased dynamic flexibility to catalytic groups. The large-loop increases the area of the interface between the subunits through its contacts and may facilitate an alternating structural cycle demanded by a half-of-sites reaction mechanism through stronger ties, as the dimer oscillates between high affinity (active) or low phosphoryl-group affinity (inactive).

Abstract P276: Tissue Primacy of Chymase as an Angiotensin II-forming Enzyme from Angiotensin-(1-12)

Hypertension ◽  
2016 ◽  
Vol 68 (suppl_1) ◽  
Author(s):  
Sarfaraz Ahmad ◽  
Jasmina Varagic ◽  
Che P Cheng ◽  
James F Collawn ◽  
Louis J Dell’Italia ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Left Ventricle ◽  
Rat Heart ◽  
Kinetic Data ◽  
Plasma Membranes ◽  
Catalytic Efficiency ◽  
Heart Tissue ◽  
Ang Ii ◽  
Efficiency Ratio

Breaking the prevailing acceptance of ACE primacy as the Ang II-forming enzyme, we have demonstrated that cardiac Ang II production in human and rat heart tissues are primarily mediated by chymase. In this study, we compared the affinity of cardiac chymase to generate Ang II from Ang-(1-12) or Ang I in plasma membranes (PMs) isolated from the diseased left atria of humans and SHR left ventricle. PMs (50-100 μg) were exposed to increasing concentrations of either Ang-(1-12) or Ang I substrate (0-300 μM) for 30 min at 37 o C in the presence of lisinopril (200 μM). The K m and V max of human cardiac chymase (Mean ± SE) were 29 ± 0.9 vs 87 ± 8.8 μM and 57 ± 1.4 vs 145 ± 3.7 μM/min/mg for Ang-(1-12) and Ang I substrates, respectively. Similarly, the K m and V max of rat cardiac chymase were 64 ± 6.3 vs 142 ± 17 μM and 13.2 ± 1.3 vs 1.9 ± 0.2 μM/min/mg for Ang-(1-12) and Ang I substrate, respectively. These data suggest that cardiac chymase has a higher affinity for Ang-(1-12) substrate compared to Ang I in both human and rat heart tissues. Further, our kinetic data show that the catalytic efficiency (ratio of V max /K m ) of human and rat chymase were 1.2 and 15.4-fold higher for Ang-(1-12) substrate compared to Ang I. Overall, our findings suggest that Ang-(1-12), rather than Ang I, is the preferred substrate for chymase in the generation of Ang II by human and rat heart tissue.

Proteomics Based Study of Soybean and Phakopsora pachyrhizi Interaction

2013 ◽  
Vol 14 (1) ◽  
pp. 29 ◽  
Author(s):  
M. Ganiger ◽  
D. R. Walker ◽  
Z. Y. Chen
Keyword(s):  
Gel Electrophoresis ◽  
Time Course ◽  
Molecular Level ◽  
Protein Profile ◽  
Differentially Expressed ◽  
Soybean Rust ◽  
Phakopsora Pachyrhizi ◽  
Yield Losses ◽  
Resistant Line ◽  
Differentially Expressed Protein

Phakopsora pachyrhizi, the causal agent of Asian soybean rust (ASR), has the potential to cause severe yield losses as all currently grown U.S. commercial soybean varieties are susceptible. In this proteomics study, we compared two soybean sibling lines, a resistant line 10G18 and a susceptible line 10G21 derived from Recombinant Inbred Line (RIL) population RN06-32-2 to understand the compatible and incompatible host-pathogen interactions at the molecular level. We compared the protein profile differences between the two lines over a time-course of 14 days with or without P. pachyrhizi inoculation using differential in-gel electrophoresis (DIGE). Approximately 70 differentially expressed spots between 10G18 and 10G21 lines with and without P. pachyrhizi inoculation were identified. Some of these spots, which were up- and down-regulated in resistant line 10G18, were sequenced using LC-MS/MS. Of the 70 differentially expressed protein spots, 31 up-regulated and 6 down-regulated spots in resistant line 10G18 were sequenced. These sequenced proteins were mostly involved in photosynthesis based on homology searches. The involvement in disease resistance for some of these differentially up-regulated proteins has been reported, indicating their possible role in soybean defense against ASR. However, further studies are necessary. Accepted for publication 3 September 2013. Published 25 November 2013.

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