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Key research themes
1. How well do in vitro enzyme kinetic constants represent in vivo catalytic rates and what methods improve this understanding?
This research area focuses on comparing enzyme kinetic constants measured in vitro, such as k_cat, with the maximal catalytic rates of enzymes inside living cells (k_vivo_max). It addresses the discrepancies caused by physiological factors like substrate saturation, thermodynamics, posttranslational modifications, molecular crowding, and metabolite concentrations which affect enzyme activity in vivo. Improving the representativeness of kinetic parameters is critical for accurate metabolic modeling and understanding cellular metabolism.
Key finding: By integrating proteomic enzyme abundance data with computed metabolic fluxes across 31 growth conditions of Escherichia coli, the study estimated in vivo maximal catalytic rates (k_vivo_max) and found a strong correlation... Read more
Key finding: The paper derived mathematical expressions extending Michaelis-Menten kinetics to conditions where enzyme concentration is comparable to substrate concentration, common in vivo but rarely addressed in vitro. The approach... Read more
2. How can thermodynamics and molecular mechanisms be integrated into enzyme kinetic models to improve understanding of reversible catalysis?
This theme explores the decomposition and extension of classical Michaelis-Menten kinetics to explicitly incorporate thermodynamic driving forces and substrate/product saturation. It addresses reversible enzyme reactions and presents separable rate laws that clarify contributions from catalytic capacity, binding saturation, and thermodynamics. Improved mechanistic models enable better quantification and prediction of enzyme behavior, especially for reversible reactions prevalent in vivo, and highlight the interplay between enzyme kinetics and thermodynamics.
Key finding: The reversible Michaelis-Menten rate law was decomposed into three multiplicative terms: catalytic capacity (maximal enzyme turnover rate), fractional substrate saturation, and thermodynamic driving force expressed via Gibbs... Read more
3. What are robust experimental and computational approaches to determine enzyme kinetic parameters and to model enzyme-catalyzed reactions accurately?
This research area focuses on the development and refinement of experimental protocols and computational models for enzyme kinetics determination, including reaction rate constants, equilibrium constants, and inhibition parameters, using advanced methodologies like isothermal titration calorimetry (ITC), nonlinear regression, and novel mathematical fitting techniques. These approaches address challenges such as instrument limitations, reversible kinetics, product inhibition, and operator bias, improving reliability and precision in parameter estimation critical for enzyme characterization and industrial applications.
Key finding: By analyzing heat rate data following a single injection of enzyme into substrate in an ITC, the authors developed methods to simultaneously determine Michaelis constant (K_M), catalytic rate constant (k_2), enthalpy change... Read more
Key finding: The paper derived new equations enabling quantitative estimation of first-order rate constants for enzyme-substrate complex activation (k+2), deactivation (k-2), and product release (k3) based on standard steady-state kinetic... Read more
Key finding: This study distinguished steady-state first-order rate constants for enzyme-substrate complex dissociation from zero-order Michaelian constants, deriving equations suitable for fitting to experimental data. It challenged the... Read more
Key finding: Presenting a quantitative framework and simple algorithm for non-dimensionalization, the paper rigorously defined the conditions underpinning the standard and reverse quasi-steady-state assumptions (QSSA) for enzyme kinetic... Read more