Mechanism of inactivation of S-adenosylhomocysteine hydrolase by (E)-5',6'-didehydro-6'-deoxy-6'-halohomoadenosines
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S-Adenosylhomocysteine (AdoHcy) hydrolase is inactivated by halogenated compounds (EDDHHAs). This study elucidates the two catalytic pathways involved in enzyme inactivation and product formation.
Area of Science:
- Biochemistry
- Enzymology
- Chemical Biology
Background:
- S-Adenosylhomocysteine (AdoHcy) hydrolase plays a crucial role in cellular metabolism.
- Irreversible inactivation of AdoHcy hydrolase by specific halogenated compounds (EDDHHAs) has been observed.
Purpose of the Study:
- To elucidate the detailed catalytic mechanisms by which EDDHHAs inactivate AdoHcy hydrolase.
- To understand the formation of adenine (Ade) and the reduction of enzyme-bound NAD+ (E.NAD+) to NADH during inactivation.
Main Methods:
- Incubation of AdoHcy hydrolase with various EDDHHAs (halogen = I, Br, Cl).
- Monitoring of halide ion and adenine release.
- Enzyme kinetics and reaction intermediate analysis.
Main Results:
- Two distinct catalytic pathways (a and b) for inactivation were identified.
- Pathway a involves water addition and halide elimination, forming 6'-carboxaldehyde, which can be oxidized to 3'-keto-6'-carboxaldehyde.
- Pathway b involves initial oxidation of EDDHHAs to 3'-keto-EDDHHAs, followed by halide release.
- Both pathways lead to the formation of adenine and inactive NADH-bound enzyme.
Conclusions:
- The inactivation mechanism involves both hydrolytic and oxidative steps.
- The rate of halide release and partition ratios correlate with enzyme inactivation efficiency.
- Understanding these pathways provides insight into enzyme inhibition and drug design.
Abstract:
S-Adenosylhomocysteine (AdoHcy) hydrolase is irreversibly inactivated by (E)-5',6'-didehydro-6'-deoxy-6'-halohomoadenosines (EDDHHAs, halogen = I, Br, and Cl). The inactivation is concomitant with the reduction of the enzyme-bound NAD+ (E.NAD+) to NADH, the release of halide ion, and the formation of adenine (Ade) from the EDDHHAs. The mechanism of this inactivation involves two catalytic pathways. Pathway a involves a rapid addition of water to the 5',6'-bond of EDDHHAs and elimination of halide ion, resulting in the formation of 6'-carboxaldehyde 1 which then degrades chemically, resulting in the formation of Ade. Alternatively, 6'-carboxyaldehyde 1 can be oxidized by E.NAD+ to form 3'-keto-6'-carboxaldehyde 3 and the NADH form (inactive) of the enzyme. Like 6'-carboxaldehyde 1, the 3'-keto derivative 3 degrades chemically to form Ade. Pathway b involves the oxidation of EDDHHAs to 3'-keto-EDDHHAs 2 by E.NAD+, as the first step, and the subsequent release of halide ion to form 3'-keto-6'-carboxaldehyde 3. Evidence in support of these mechanisms includes the observations that incubation of EDDHHAs with AdoHcy hydrolase generated large molar excesses of halide ions and Ade, that Ade was shown to eliminate spontaneously from 6'-carboxaldehyde 1, and that the more rapid the halide ion release (Cl- > Br- > I-) from the EDDHHAs or the greater the partition ratios (nonlethal turnovers/lethal event), the lower the enzyme inactivation efficiency.(ABSTRACT TRUNCATED AT 250 WORDS)