Enzyme Kinetic Profiling Service

Enzyme Kinetic Profiling is an indispensable biochemical service that quantitatively measures the activity and efficiency of an enzyme under defined conditions. This service determines key kinetic parameters such as Km (Michaelis constant), Vmax (maximum reaction velocity), kcat (turnover number), and the specificity constant (kcat/Km). By analyzing the reaction rate over a range of substrate concentrations, we provide a precise mathematical model of the enzyme's function.

CD Biosynsis offers a comprehensive CRO service for Enzyme Kinetic Profiling, essential for drug discovery, enzyme engineering, and mechanistic studies. We utilize high-throughput assays, sophisticated spectral detection methods (e.g., UV-Vis, Fluorescence, Luminescence), and rigorous non-linear regression analysis to ensure high-quality and reliable data. Our platform is adaptable to diverse enzyme classes, including oxidoreductases, transferases, hydrolases, and lyases, delivering actionable insights into substrate specificity, inhibitor potency, and optimal reaction environments.

Highlights

Our service provides high-resolution kinetic data crucial for characterizing enzyme function and screening potential modulators.

• Accurate Parameter Determination: Precise measurement of Km, Vmax, kcat, and specificity constant (kcat/Km) using established models (Michaelis-Menten, Hill).
• Inhibitor Screening and Analysis: Determine inhibitor potency (IC50) and binding affinity (Ki) for various mechanisms (competitive, uncompetitive, non-competitive).
• Substrate Specificity Profiling: Compare the catalytic efficiency of an enzyme against a panel of potential substrates.
• Assay Development and Optimization: Customized assay design for novel enzymes or non-standard reaction conditions.

Applications

Kinetic profiling is fundamental for quantifying enzyme behavior, driving progress in various R&D areas:

Drug Target Validation & Screening

           

Quantification of the efficacy of lead compounds by determining inhibition constants (Ki) against therapeutic targets.

Enzyme Engineering and Mutagenesis

Characterizing the functional impact of point mutations to improve stability, activity, or alter substrate promiscuity for biocatalysis.

Biocatalyst Optimization

Identifying optimal pH, temperature, and cofactor concentrations for industrial enzyme applications to maximize yield and efficiency.

Mechanistic Biochemistry Studies

Elucidating complex reaction pathways, identifying steady-state intermediates, and performing full kinetic mechanism analysis.

Platform

Our Enzyme Kinetic Profiling platform utilizes state-of-the-art instrumentation and rigorous data analysis protocols.

High-Throughput Screening (HTS)

Automated liquid handling and plate readers for rapid and efficient measurement of hundreds of kinetic data points simultaneously.

Advanced Detection Methods

Including absorbance (UV-Vis), fluorescence intensity, time-resolved fluorescence (TRF), and chemiluminescence.

Isothermal Titration Calorimetry (ITC)

For label-free, direct measurement of thermodynamic parameters (Delta H, Delta S) and binding affinity (Kd) to complement kinetic data.

Pre-Steady-State Kinetics

Utilizing stopped-flow systems for characterizing rapid reaction transients, essential for complex mechanism study.

Non-Linear Regression Analysis

Utilizing professional software for fitting kinetic models, ensuring accurate parameter estimation and statistical validation.

Workflow

Our Enzyme Kinetic Profiling process is structured to deliver accurate, reproducible, and comprehensive kinetic datasets:

• Assay Establishment and Optimization: Define optimal buffer, pH, temperature, and enzyme concentration to ensure a robust, linear initial velocity phase.
• Substrate Titration Experiment: Measure initial reaction velocity (v0) across a broad range of substrate concentrations ([S]).
• Inhibitor/Modulator Testing (Optional): Measure v0 in the presence of varying concentrations of test compounds and substrates.
• Kinetic Data Analysis: Plot v0 vs. [S] and perform non-linear curve fitting to the Michaelis-Menten model or other appropriate kinetic models.
• Reporting: Deliver a comprehensive report including all raw data, fitted curves, calculated Km, Vmax, kcat, and inhibition constants (Ki) with statistical confidence limits.

CD Biosynsis guarantees statistically validated kinetic parameters and expert mechanistic interpretation. Every project includes:

• Calculated Kinetic Constants: Km, Vmax, kcat with standard errors.
• Full Inhibition Profile: IC50 and Ki values for tested compounds, including the predicted inhibition mechanism.
• Raw Data and Fitted Plots: All measured data points and high-quality graphics of the fitted kinetic models.
• Detailed Protocol: A reproducible method for the established enzymatic assay.