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Underrepresented Impurities in 4-Hydroxyphenylpyruvate Affect the Catalytic Activity of Multiple Enzymes

Andrew Parkins¹, Suzanne I Sandin^2,3, Jonathon Knittel¹

¹Department of Chemistry, University of the Pacific, Stockton, California 95211, United States.

Analytical Chemistry

|March 6, 2023

Abstract:

Macrophage migration inhibitory factor (MIF) is a key immunostimulatory protein with regulatory properties in several disorders, including inflammation and cancer. All the reported inhibitors that target the biological activities of MIF have been discovered by testing against its keto/enol tautomerase activity. While the natural substrate is still unknown, model MIF substrates are used for kinetic experiments. The most extensively used model substrate is 4-hydroxyphenyl pyruvate (4-HPP), a naturally occurring intermediate of tyrosine metabolism. Here, we examine the impact of 4-HPP impurities in the precise and reproducible determination of MIF kinetic data. To provide unbiased evaluation, we utilized 4-HPP powders from five different manufacturers. Biochemical and biophysical analyses showed that the enzymatic activity of MIF is highly influenced by underrepresented impurities found in 4-HPP. Besides providing inconsistent turnover results, the 4-HPP impurities also influence the accurate calculation of ISO-1's inhibition constant, an MIF inhibitor that is broadly used for in vitro and in vivo studies. The macromolecular NMR data show that 4-HPP samples from different manufacturers result in differential chemical shift perturbations of amino acids in MIF's active site. Our MIF-based conclusions were independently evaluated and confirmed by 4-hydroxyphenylpyruvate dioxygenase (HPPD) and D-dopachrome tautomerase (D-DT); two additional enzymes that utilize 4-HPP as a substrate. Collectively, these results explain inconsistencies in previously reported inhibition values, highlight the effect of impurities on the accurate determination of kinetic parameters, and serve as a tool for designing error-free in vitro and in vivo experiments.

Insights

Impurities in 4-hydroxyphenyl pyruvate (4-HPP), a model substrate for macrophage migration inhibitory factor (MIF), significantly impact kinetic data. This affects the accurate determination of MIF inhibitor constants, crucial for drug development.

Area of Science:

Biochemistry
Enzymology
Drug Discovery

Background:

Macrophage migration inhibitory factor (MIF) is a critical protein in immune regulation, inflammation, and cancer.
Current inhibitors targeting MIF's keto/enol tautomerase activity are developed using model substrates like 4-hydroxyphenyl pyruvate (4-HPP).
The precise kinetic characterization of MIF and its inhibitors is essential for therapeutic development.

Purpose of the Study:

To investigate the impact of impurities in 4-hydroxyphenyl pyruvate (4-HPP) on the kinetic analysis of macrophage migration inhibitory factor (MIF).
To evaluate how 4-HPP impurities affect the determination of inhibition constants for MIF inhibitors, such as ISO-1.
To provide a basis for more accurate and reproducible in vitro and in vivo experimental designs.

Main Methods:

Biochemical and biophysical analyses of MIF activity using 4-HPP from five different manufacturers.
Determination of inhibition constants for MIF inhibitors.
Macromolecular Nuclear Magnetic Resonance (NMR) spectroscopy to study MIF-4-HPP interactions.
Independent validation using other 4-HPP-utilizing enzymes: 4-hydroxyphenylpyruvate dioxygenase (HPPD) and D-dopachrome tautomerase (D-DT).

Main Results:

Underrepresented impurities in 4-HPP significantly influence MIF's enzymatic activity and kinetic parameters.
Inconsistent turnover results and inaccurate calculation of inhibition constants were observed due to 4-HPP impurities.
NMR data revealed differential interactions between MIF's active site and 4-HPP samples from various sources.
Findings were corroborated by experiments with HPPD and D-DT, confirming the broad impact of 4-HPP quality.

Conclusions:

The purity of 4-HPP is critical for the accurate and reproducible determination of MIF kinetic data and inhibitor potency.
Previously reported inconsistencies in MIF inhibition values may be attributed to variations in 4-HPP purity.
These findings underscore the importance of quality control for substrates in biochemical assays and guide the design of reliable in vitro and in vivo studies.

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