Reaction Chemical Kinetics in Biology

Harmer, Nicholas J.; Vega, Mirella Vivoli

doi:10.1002/9781119483977.ch9

Cited by 4 publications

(4 citation statements)

References 66 publications

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“…Mechanistic modeling has been demonstrated as an effective method for optimizing and understanding a number of multienzyme systems. − In many fields, ensemble modeling is increasingly employed to acknowledge uncertainty in parameters of mechanistic or deterministic models. − Importantly, this allows the use of distributions to describe a plausible set of values for each parameter, which is preferable to methods that seek to fit a single set of parameters to the entire model. − However, despite recent successes in the use of mechanistic models for reaction optimization, models often require simplifications or assumptions which result in incorrect predictions where these assumptions are no longer valid. For example, the Michaelis–Menten equation requires the assumption that the enzyme–substrate complex is at steady state …”

Section: Introductionmentioning

confidence: 99%

“…For example, the Michaelis−Menten equation requires the assumption that the enzyme−substrate complex is at steady state. 32 An alternative approach is the use of empirical modeling, often employed using design of experiments (DoE). 33 Particularly where systems are too prohibitively complex for mechanistic modeling, empirical modeling offers a powerful solution for understanding how process inputs affect process outputs, albeit without mechanistic understanding.…”

Section: ■ Introductionmentioning

confidence: 99%

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Rapid Model-Based Optimization of a Two-Enzyme System for Continuous Reductive Amination in Flow

Finnigan

Citoler

Cosgrove

et al. 2020

Org. Process Res. Dev.

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Enzymes are increasingly combined into multienzyme systems for cost and productivity benefits. Further advantages can be gained through the use of immobilized enzymes, allowing continuous biotransformations in flow. However, the optimization of such multienzyme systems is challenging, particularly where immobilized enzymes are used. Here, we meet this challenge using both mechanistic and empirical modeling to optimize a reaction involving a reductive aminase and a glucose dehydrogenase for continuous biocatalytic reductive amination in flow. Crucially, the construction of the mechanistic model was achieved quickly, with only a few important parameters determined experimentally, and ensemble modeling used to facilitate the use of estimates or literature values. Upon reaching the limits of the mechanistic model's capabilities, we show that solution space can be further explored using a definitive screening design to generate an empirical model of the reaction, using the mechanistic model's prediction as a starting point. We demonstrate the use of this empirical model to design optimal processes for either high productivity or to minimize necessary cofactor and cosubstrate concentrations. Our results demonstrate that the synergistic use of both mechanistic and empirical modeling offers a route for rapid optimization of multienzyme systems of immobilized enzymes in flow with minimal experimental effort.

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Section: Introductionmentioning

confidence: 99%

Section: ■ Introductionmentioning

confidence: 99%

Rapid Model-Based Optimization of a Two-Enzyme System for Continuous Reductive Amination in Flow

Finnigan

Citoler

Cosgrove

et al. 2020

Org. Process Res. Dev.

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“…Two experimental replicates were taken for each data point. Data were fitted to the sequential bi-bi and ping-pong Equations (1) , (2) in Prism 9.0.1 (GraphPad) ( 73 , 74 , 75 ). To determine the effect of divalent cations, the coupling enzymes were first tested in a mixture of 40 mM Hepes, pH 7.5, 100 mM KCl, 5 mM DTT, 100 μg/ml BSA, 200 μM NADH, 500 μM phosphoenolpyruvate, 1 mM ADP, and 0.2 U/ml PK-LD.…”

Section: Methodsmentioning

confidence: 99%

The ROK kinase N-acetylglucosamine kinase uses a sequential random enzyme mechanism with successive conformational changes upon each substrate binding

Roy¹,

Vega²,

Ames³

et al. 2023

Journal of Biological Chemistry

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“…Two experimental replicates were taken for each data point. Data were fitted to the sequential bi-bi and ping-pong equations in Prism 9.01 (GraphPad) (62)(63)(64). To determine the effect of divalent cations, initial reaction rates were determined with the MgCl2 in the mixture above substituted with of 10 mM MgCl2, MnCl2, CaCl2, CuCl2, or CoCl2, and normalized to the rate with MgCl2.…”

Section: Methodsmentioning

confidence: 99%

Structure and function of N-acetylglucosamine kinase illuminates the catalytic mechanism of ROK kinases

Roy

Ames

et al. 2021

Preprint

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N-acetyl-D-glucosamine (GlcNAc) is a major component of bacterial cell walls. Many organisms recycle GlcNAc from the cell wall or metabolise environmental GlcNAc. The first step in GlcNAc metabolism is phosphorylation to GlcNAc-6-phosphate. In bacteria, the ROK family kinase NagK performs this activity. Although ROK kinases have been studied extensively, no ternary complex showing the two substrates has yet been observed. Here, we solved the structure of NagK from the human pathogen Plesiomonas shigelloides in complex with GlcNAc and the ATP analogue AMP-PNP. Surprisingly, PsNagK showed two conformational changes associated with the binding of each substrate. Consistent with this, the enzyme showed a sequential random enzyme mechanism. This indicates that the enzyme acts as a coordinated unit responding to each interaction. Molecular dynamics modelling of catalytic ion binding confirmed the location of the essential catalytic metal. Site-directed mutagenesis confirmed the catalytic base, and that the metal coordinating residue is essential. Together, this study provides the most comprehensive insight into the activity of a ROK kinase.

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Reaction Chemical Kinetics in Biology

Cited by 4 publications

References 66 publications

Rapid Model-Based Optimization of a Two-Enzyme System for Continuous Reductive Amination in Flow

Rapid Model-Based Optimization of a Two-Enzyme System for Continuous Reductive Amination in Flow

The ROK kinase N-acetylglucosamine kinase uses a sequential random enzyme mechanism with successive conformational changes upon each substrate binding

Structure and function of N-acetylglucosamine kinase illuminates the catalytic mechanism of ROK kinases

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