Active-Site Probe Labeling of Enzymes

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Active-site probe labeling is a powerful technique for selectively modifying enzymes at their catalytic centers to investigate mechanism, activity, structure, and molecular interactions. Through the use of highly specific chemical probes, this method allows direct interrogation of the active site environment, enabling researchers to monitor conformational changes, characterize catalytic residues, and quantify enzyme activity with exceptional precision. Creative Enzymes provides comprehensive, custom-built active-site labeling services that integrate advanced probe chemistry, structural analysis, and rigorous quality control. Our scientific team supports clients across research, biotechnology, diagnostics, and industrial biocatalysis by designing and executing tailored labeling workflows that preserve enzyme integrity while delivering precise, reproducible, and application-ready results.

Introduction to Enzyme Active-Site Probe Labeling

Enzyme function depends fundamentally on the structure, composition, and microenvironment of the active site—the region where substrate binding, catalysis, and turnover occur. Modifying this region selectively offers a unique window into mechanistic behavior and catalytic efficiency. However, the active site is often deeply recessed, structurally dynamic, and surrounded by critical residues that must remain intact for function. As a result, active-site labeling requires highly selective probes and carefully controlled reaction conditions to avoid compromising activity.

Figure 1. An example of enzyme active-site probe labeling: Click chemistry-based labeling of serine hydrolases. The active enzyme is allowed to react with the 1-alkyne phosphonate derivative. Then, the tag, in this case an azido derivative of fluorescein, is added to form the final adduct. (Sotiropoulou et al., 2022)

Active-site probe labeling of enzymes has become an essential tool in biochemistry and biotechnology for purposes such as:

Tracking catalytic turnover through fluorescent, radiolabeled, or affinity-based probes
Identifying active populations in complex mixtures
Investigating conformational dynamics and substrate specificity
Distinguishing functional isozymes or variants
Studying inhibitor binding and structure–activity relationships
Developing activity-based assays or diagnostic platforms

Given the complexity of enzyme structures and the diversity of available probe chemistries, successful active-site labeling depends on deep enzymological expertise, rational design, and meticulous optimization. Creative Enzymes brings together decades of experience in enzymology, chemical modification, and protein engineering to deliver precise, targeted labeling solutions that maintain biological function while providing clear analytical value.

Enzyme Active-Site Probe Labeling: What We Offer

Creative Enzymes provides a full suite of customized active-site probe labeling of enzymes services designed to support mechanistic research, assay development, and industrial biocatalysis. Our offerings include:

Services
Probe Selection, Design, and Custom Synthesis	We identify or design probes with appropriate specificity, reactivity, and detection modality. Available probe types include: Reactive covalent modifiers (e.g., fluorophosphonates, iodoacetamides, sulfonyl fluorides) Substrate analogs with reporter groups Mechanism-based inhibitors Photoreactive probes Fluorescent, biotinylated, or isotopically labeled probes	Get a quote
Site-Specific and Mechanism-Guided Labeling	We perform highly controlled labeling reactions that selectively target catalytic residues without damaging structural integrity.
Structural and Functional Characterization	Comprehensive analyses confirm labeling efficiency, residue specificity, and preserved catalytic activity. Typical QC methods include: Mass spectrometry SDS-PAGE and Western blot Fluorescence or absorbance quantification Activity assays Peptide mapping
Purification and Recovery of Labeled Enzymes	Purified products are provided in a stable, buffer-optimized format suitable for downstream use in research, diagnostics, or industrial processes.
Large-Scale and Reproducible Production	We support milligram- to gram-scale production for commercial or industrial applications, ensuring batch-to-batch consistency and strict process control.

Service Workflow

Workflow of enzyme active-site probe labeling service

Scale and Throughput

We support:

Discovery-scale (50 µg–1 mg) for mechanistic studies
Assay-development scale (1–20 mg)
Production scale (>50 mg to multi-gram) for commercial use

Delivery Format

Labeled enzymes can be supplied:

Lyophilized
In activity-preserving cryoprotectant buffers
In custom formulations for specific applications (e.g., immobilization-ready)

Contact Our Team

Why Choose Us

Exceptional Enzymology Expertise

Our team consists of specialists in catalytic mechanisms, structural chemistry, and protein modification, ensuring nuanced control over active-site labeling.

Advanced Probe Chemistry Capabilities

We utilize a diverse library of reactive probes and can design custom solutions for complex or rare enzymatic targets.

Accurate, Reproducible, and Scalable Processes

Our optimized workflows ensure consistent results across research-scale and large-scale production.

Comprehensive Analytical Characterization

Every project includes rigorous QC to verify labeling efficiency, residue specificity, purity, and catalytic performance.

Tailored Strategies for Sensitive or Difficult Enzymes

We excel at labeling enzymes with challenging structural or chemical requirements, preserving function while achieving selective modification.

Reliable Customer Support and Transparent Communication

Clients benefit from clear documentation, regular updates, and expert guidance throughout the project.

Enzyme Active-Site Probe Labeling: Case Studies

Case 1: Active-Site Directed Probes to Report Enzymatic Action in UPS

Irreversible covalent inhibitors with reporter groups, known as activity-based probes, enable the study of enzymes based on catalytic activity rather than expression levels, providing more accurate insights into protein function and cellular consequences. These probes can be applied directly to cells or tissues and are suitable for molecular imaging and pharmacological studies. The discussion focuses on probes targeting enzymatic activities within the ubiquitin–proteasome system, a pathway critical for protein regulation and a promising target for anticancer therapy. Such probes facilitate functional analysis and may accelerate the development of targeted therapeutic strategies.

Active-site directed probes to report enzymatic action in the ubiquitin proteasome system Figure 2. Applications of mechanism-based active-site directed probes for detection of proteasome and DUB activity. (Ovaa, 2007)

Case 2: Activity-Based Probes for Monitoring Sulfoquinovosidase Function

Sulfoquinovosyl diacylglycerol (SQDG) is a major sulfur reservoir in nature, and its microbial degradation begins with sulfoquinovosidases (SQases), enzymes that release sulfoquinovose from SQDG. To study these GH31-family enzymes in complex environments, researchers developed SQ cyclophellitol-aziridine activity-based probes (ABPs). These probes covalently label the active-site nucleophile, allowing selective detection and visualization of SQases. A Cy5-labeled probe enables fluorescence imaging in crude lysates and monitoring of SQase activity during bacterial growth, while a biotinylated probe supports affinity capture and proteomic identification. These tools provide powerful new capabilities for investigating SQases in microbial ecology and human microbiota.

Detection of sulfoquinovosidase activity in cell lysates using activity‐based probes Figure 3. The development of activity-based probes targeting the active site nucleophile of glycoside hydrolase family 31 sulfoquinovosidases. The probes are shown to bind covalently by structural biology, mass spectrometry and Cy5 in-gel fluorescence, are selective for sulfoquinovosidases in whole bacterial cell lysates, and are used to monitor active levels of enzyme over time. (Li et al., 2024)

Enzyme Active-Site Probe Labeling: FAQs

Q: Does active-site labeling affect enzyme activity?

A: Yes—by design. Active-site probes typically bind to or modify catalytic residues. However, our strategies minimize unwanted structural disruptions and ensure the labeled enzyme remains suitable for the intended application.
Q: What types of probes can you work with?

A: We support covalent inhibitors, substrate analogs, fluorogenic and chromogenic probes, affinity reporters, photoreactive groups, and custom-synthesized reagents.
Q: Can you label enzymes that are unstable or prone to aggregation?

A: Yes. We optimize buffers, reaction temperatures, and probe concentrations to maintain stability. Mild chemistries are available when needed.
Q: Do you support site-specific mapping of labeled residues?

A: Absolutely. We offer peptide mapping, mass spectrometry, and structural interpretation to identify precise modification sites.
Q: Is it possible to scale the labeling process for commercial applications?

A: Yes—our workflows are fully scalable, from microgram quantities to multi-gram production.
Q: How do you confirm labeling selectivity?

A: We combine structural prediction with mass spectrometry, activity assays, and probe-specific detection methods to verify specificity and efficiency.
Q: What information is required to start a project?

A: We request the enzyme source, purity, sequence (if available), buffer composition, intended application, stability data, and any structural information that may inform probe selection.
Q: Do you provide method transfer or long-term production support?

A: Yes. We offer process transfer, ongoing batch production, and documentation to ensure long-term reproducibility.

References:

Li Z, Pickles IB, Sharma M, et al. Detection of sulfoquinovosidase activity in cell lysates using activity‐based probes. Angewandte Chemie. 2024;136(26):e202401358. doi:10.1002/ange.202401358
Ovaa H. Active-site directed probes to report enzymatic action in the ubiquitin proteasome system. Nat Rev Cancer. 2007;7(8):613-620. doi:10.1038/nrc2128
Sotiropoulou G, Zingkou E, Bisyris E, Pampalakis G. Activity-based probes for proteases pave the way to theranostic applications. Pharmaceutics. 2022;14(5):977. doi:10.3390/pharmaceutics14050977

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Project Description:

For research and industrial use only. Not intended for personal medicinal use. Certain food-grade products are suitable for formulation development in food and related applications.

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