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Inhibitors of abscisic acid 8'-hydroxylase
A J Cutler1, P A Rose, T M Squires
1Plant Biotechnology Institute, National Research Council of Canada, 110, Gymnasium Place, Saskatoon, Saskatchewan S7N 0W9, Canada. adrian.cutler@nrc.ca
Biochemistry
|November 7, 2000
Summary
New abscisic acid (ABA) analogues act as suicide substrates and competitive inhibitors for ABA 8'-hydroxylase, impacting plant hormone metabolism and seed germination. These compounds show enhanced hormonal activity in physiological assays.
Area of Science:
- Plant biochemistry
- Hormone metabolism
- Enzyme inhibition
Background:
- (+)-Abscisic acid (ABA) is a key plant hormone regulating various physiological processes.
- ABA catabolism, primarily through 8'-hydroxylation, is crucial for controlling ABA levels.
- ABA 8'-hydroxylase is the enzyme responsible for this critical catabolic step.
Purpose of the Study:
- To synthesize and evaluate structural analogues of (+)-ABA as potential inhibitors of (+)-ABA 8'-hydroxylase.
- To investigate the mechanism of inhibition, distinguishing between suicide substrates and competitive inhibitors.
- To explore the physiological relevance of these inhibitors in plant processes like seed germination.
Main Methods:
- Synthesis of novel (+)-ABA structural analogues.
- Enzyme assays using microsomes from suspension-cultured corn cells to measure ABA 8'-hydroxylase activity.
- Characterization of inhibition kinetics (suicide substrate vs. competitive inhibition).
- Assay of inhibition of Arabidopsis thaliana seed germination.
Main Results:
- Four ABA analogues [(+)-8'-acetylene-ABA, (+)-9'-propargyl-ABA, (-)-9'-propargyl-ABA, and (+)-9'-allyl-ABA] were identified as suicide substrates for ABA 8'-hydroxylase.
- (+)-9'-propargyl-ABA was the most effective suicide substrate (K(I) = 0.27 microM).
- Several analogues, notably (+)-8'-propargyl-ABA (K(i) = 1.1 microM), acted as competitive inhibitors.
- The 7'-hydroxylation of (-)-ABA by a different enzyme was not inhibited by these suicide substrates.
- (+)-9'-propargyl-ABA and (+)-8'-acetylene-ABA demonstrated enhanced hormonal activity in inhibiting Arabidopsis thaliana seed germination compared to (+)-ABA.
Conclusions:
- Novel ABA analogues can effectively inhibit ABA 8'-hydroxylase through suicide substrate and competitive inhibition mechanisms.
- These findings provide insights into the enzyme's active site and substrate positioning.
- The synthesized analogues, particularly (+)-9'-propargyl-ABA and (+)-8'-acetylene-ABA, represent potent tools for manipulating ABA levels and exhibit significant physiological activity, offering potential for agricultural applications.
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