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Active site generation of a protonically unstable suicide substrate from a stable precursor: glucose oxidase and
D J Porter1, J G Voet, H J Bright
1Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA. djp39807@glaxowellcome.com
Abstract:
Bromonitromethane is an inefficient suicide substrate for glucose oxidase (in contrast to the case of CH(3)CCl=NO(2)(-) and D-amino acid oxidase) because, in the enzyme-substrate encounter step, the required ionization states of enzyme (EH(0)(+), pK(a) approximately 3.5) and substrate (CHBr=NO(2)(-), pK(a) approximately 8.3) cannot be highly populated simultaneously. Because reprotonation of CHBr=NO(2)(-) is rapid at the pH value used for the assay of glucose oxidase, presentation of the enzyme with the preformed anion could not be exploited in this case. We circumvent this difficulty by allowing the enzyme to reductively dehalogenate CHBr(2)NO(2), thereby generating the desired protonically unstable suicide substrate in situ (E(r) + CHBr(2)NO(2) --> E(o) + CHBr=NO(2)(-) + HBr + H(+)). Irreversible inactivation of the enzyme, because of the formation of a dead-end N-5 formylflavin adduct, is more than 100-fold faster when CHBr=NO(2)(-) is generated in situ than when it is externally applied. The remaining competitive fates of CHBr=NO(2)(-) at the active site are protonation and release or oxidation to HCOBr (or HCONO(2)). Strong support for these conclusions comes from (1) the brisk evolution of CH(3)CBr=NO(2)(-) (which is too bulky to act further as an efficient suicide substrate) from the enzyme-catalyzed reductive debromination of CH(3)CBr(2)NO(2), (2) the 1:1 stoichiometry of enzyme inactivation, and (3) the identification of the modified flavin as 5-formyl-1, 5-dihydro-FAD.
Insights
Bromonitromethane is a poor suicide substrate for glucose oxidase due to ionization issues. Generating the substrate in situ significantly enhances enzyme inactivation, overcoming these limitations for improved research applications.
Area of Science:
- Biochemistry
- Enzyme kinetics
- Chemical biology
Background:
- Bromonitromethane is an inefficient suicide substrate for glucose oxidase.
- Simultaneous ionization of enzyme and substrate is difficult at physiological pH.
- Reprotonation of the bromonitromethane anion is rapid, preventing in situ generation via preformed anion.
Purpose of the Study:
- To overcome the inefficient substrate ionization for glucose oxidase.
- To develop a method for generating the unstable bromonitromethane anion in situ.
- To enhance the rate of enzyme inactivation by a suicide substrate.
Main Methods:
- Enzyme-catalyzed reductive dehalogenation of dibromonitromethane to generate bromonitromethane anion in situ.
- Assaying glucose oxidase activity and inactivation rates.
- Spectroscopic identification of modified flavin adducts.
Main Results:
- In situ generation of bromonitromethane anion leads to >100-fold faster enzyme inactivation compared to external application.
- Reductive debromination of a related compound produced a bulky analog, supporting the mechanism.
- A 1:1 stoichiometry of enzyme inactivation was observed.
- The modified flavin was identified as a 5-formyl-1, 5-dihydro-FAD adduct.
Conclusions:
- In situ generation of bromonitromethane anion is an effective strategy to overcome ionization challenges.
- The mechanism involves reductive dehalogenation followed by irreversible enzyme inactivation.
- This approach significantly enhances the utility of bromonitromethane as a suicide substrate for glucose oxidase.