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Transition-state structures for enzymatic and alkaline phosphotriester hydrolysis
S R Caldwell1, F M Raushel, P M Weiss
1Department of Chemistry, Texas A&M University, College Station 77843.
Biochemistry
|July 30, 1991
Summary
Investigating oxygen-18 isotope effects in phosphotriester hydrolysis reveals insights into reaction mechanisms. Alkaline and enzymatic hydrolysis pathways show distinct transition states, with enzymatic hydrolysis of one substrate indicating a more advanced transition state.
Area of Science:
- Biochemistry
- Chemical Kinetics
- Isotope Effects
Background:
- Phosphotriesters are crucial in biological systems and chemical synthesis.
- Understanding their hydrolysis mechanisms is key to enzyme function and reaction design.
- Isotope effects provide sensitive probes of transition state structures.
Purpose of the Study:
- To elucidate the hydrolysis mechanisms of two phosphotriesters (I and II) under alkaline and enzymatic conditions.
- To determine the transition state structures using primary and secondary oxygen-18 (¹⁸O) isotope effects.
- To compare the mechanistic pathways of alkaline and phosphotriesterase-catalyzed hydrolysis.
Main Methods:
- Synthesis of isotopically labeled phosphotriesters ([¹⁵N, phosphoryl-¹⁸O]-, [¹⁵N, phenolic-¹⁸O]-, and [¹⁵N]-) for precise kinetic studies.
- Measurement of primary and secondary ¹⁸O kinetic isotope effects for alkaline (KOH) hydrolysis.
- Measurement of primary and secondary ¹⁸O kinetic isotope effects for enzymatic hydrolysis catalyzed by phosphotriesterase.
Main Results:
- Alkaline hydrolysis of both compounds I and II exhibited significant ¹⁸O isotope effects, consistent with an associative, SN2-like mechanism and a rate-limiting hydroxide addition.
- Enzymatic hydrolysis of compound I showed minimal ¹⁸O isotope effects, indicating the chemical step is not rate-limiting.
- Enzymatic hydrolysis of compound II displayed substantial ¹⁸O isotope effects comparable to alkaline hydrolysis, suggesting a rate-limiting chemical step and a more progressed transition state in the enzymatic reaction.
Conclusions:
- The ¹⁸O isotope effects support an associative mechanism for both alkaline and enzymatic hydrolysis, with varying degrees of bond order changes in the transition state.
- The enzymatic hydrolysis of compound II proceeds via a rate-limiting chemical step with a transition state structure distinct from alkaline hydrolysis.
- The findings provide evidence against a stable phosphorane intermediate in these hydrolysis reactions.