Enzyme Substrate Profiling Service

Enzyme Substrate Profiling is a systematic biochemical service designed to comprehensively analyze an enzyme's catalytic promiscuity and specificity against a diverse panel of potential substrates. This process identifies the enzyme's preferred natural and non-natural substrates, characterizes its relative catalytic efficiency (kcat/Km) for each, and maps the structural determinants of substrate recognition. This deep functional understanding is crucial for industrial biocatalyst design, pathway mapping, and toxicology studies.

CD Biosynsis delivers a specialized CRO service for Substrate Profiling, leveraging customized, high-throughput screening libraries that can span hundreds of structurally diverse compounds, including metabolites, small molecules, and synthetic analogues. Our platform integrates automated liquid handling, sensitive kinetic measurements, and advanced bioinformatics to provide quantitative data on substrate acceptance range, turnover rate, and binding affinity. We help clients transform an enzyme's unknown function into a precise, validated substrate profile, accelerating discovery and engineering projects.

Highlights

Our platform systematically characterizes an enzyme's substrate repertoire, providing actionable data for functional enhancement and mechanistic studies.

• Quantitative Specificity Constant: Measure and compare the specificity constant (kcat/Km) for a full library of substrates to define preference hierarchy.
• Broad Substrate Library Design: Customized or pre-designed libraries covering structurally diverse metabolites, synthetic probes, and lead compounds.
• Promiscuity and Off-Target Mapping: Identify non-natural substrates to assess catalytic promiscuity or potential off-target effects in drug metabolism.
• High-Throughput Screening (HTS): Utilize automated systems for rapid screening and profiling of large substrate sets.

Applications

Substrate profiling is a cornerstone for functional genomics, enzyme modification, and preclinical research:

Biocatalyst Optimization

           

Identifying the most efficient non-natural substrates for industrial enzymes and tailoring their specificity through directed evolution.

Metabolic Pathway Elucidation

Mapping the precise biochemical role of novel enzymes by linking them to their true endogenous substrates in a pathway.

Drug Metabolism and Toxicology

Profiling drug-metabolizing enzymes (e.g., Cytochrome P450s) against drug candidates to predict metabolic clearance and potential adverse interactions.

Enzyme Functional Annotation

Validating the predicted function of uncharacterized proteins identified through genomic sequencing projects.

Platform

Our Substrate Profiling platform integrates combinatorial chemistry, robotics, and advanced detection technologies.

Custom Substrate Library Synthesis

Synthesis of tailor-made compound libraries focusing on specific structural motifs, charge, or stereochemistry relevant to the enzyme class.

LC-MS/MS Based Detection

Utilization of highly sensitive mass spectrometry for label-free detection and quantification of reaction products, essential for native substrates.

Fluorescent and Colorimetric Assays

High-throughput screening using quick, sensitive reporter substrates for initial activity identification.

Enzyme Kinetic Characterization

Detailed determination of Km, Vmax, and kcat/Km for the best-performing substrates identified in the primary screen.

Bioinformatics and Chemogenomics

Structural analysis of active substrates to generate pharmacophore models and predict structural features required for binding.

Workflow

Our Substrate Profiling workflow is a two-phase process, moving from high-throughput screening to quantitative characterization:

• Substrate Library Selection/Design: Define the scope (e.g., metabolite set, chemical class) and source or synthesize the compound library.
• Primary Activity Screening (HTS): Incubate the enzyme with all library compounds and measure initial reaction velocity (v0) under standard conditions to identify "hits."
• Secondary Validation and Quantification: Confirm activity for the hit compounds and determine their Km and kcat/Km values in detail.
• Data Analysis and Structure-Activity Relationship (SAR) Mapping: Analyze the kinetic data, plot the relative efficiencies, and deduce the structural features responsible for specificity.
• Reporting: Deliver a ranked list of active substrates, their calculated kinetic parameters, and a detailed SAR report with recommendations for enzyme engineering or functional assignment.

CD Biosynsis guarantees a precise, quantitative assessment of enzyme specificity, providing direct guidance for your R&D programs. Every project includes:

• Ranked Substrate List: A quantitative list of all tested compounds ranked by kcat/Km efficiency.
• Full Kinetic Parameters: Km, Vmax, and kcat values for the top active substrates.
• SAR Analysis Report: Insight into the structural motifs of the substrate that govern catalytic activity and binding.
• Detailed HTS Data: All raw and processed data from the primary screening experiments.