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Inactivation of pyridoxal phosphate dependent enzymes by mono- and polyhaloalanines
Abstract:
beta,beta-Dichloro- and beta,beta,beta-trifluoroalanine irreversibly inactivate a number of pyridoxal phosphate dependent enzymes which catalyze beta- or gamma-elimination reactions. The inactivation is time dependent and the rate of inactivation is first order in enzyme concentration. This suggests that inactivation is due to covalent modification of the enzyme by a species generated at the active site from the polyhaloalanine (i.e., suicide inactivation). Monohaloalanines are substrates and do not inactivate. For gamma-cystathionase, covalent and stoichiometric attachment of [1-14C]beta,beta,beta-trifluoroalanine was shown. It is proposed that the mechanism of inactivation involves Schiff base formation between inactivator and enzyme-bound pyridoxal and subsequent elimination of HC1 from dichloroalanine or HF from trifluoroalanine. This results in the formation of a beta-halo-alpha,beta unsaturated imine, an activated Michael acceptor. Michael addition of a nucleophile at the active site leads to covalent labeling of the enzyme and inactivation. Alanine racemase is also inactivated by the two polyhaloalanines. Glutamate-pyruvate and gultamate-oxaloacetate transaminase are inactivated by monohaloalanines but not by polyhaloalanines.
Insights
Polyhaloalanines irreversibly inactivate pyridoxal phosphate-dependent enzymes through suicide inactivation. This mechanism involves covalent modification of the enzyme
Area of Science:
- Biochemistry
- Enzyme kinetics
- Organic chemistry
Background:
- Pyridoxal phosphate (PLP)-dependent enzymes catalyze crucial beta- or gamma-elimination reactions.
- Polyhaloalanines are analogs of amino acids that can potentially interact with PLP-dependent enzymes.
Purpose of the Study:
- To investigate the mechanism by which beta,beta-dichloro- and beta,beta,beta-trifluoroalanine inactivate PLP-dependent enzymes.
- To determine the specificity of polyhaloalanines and monohaloalanines as enzyme inactivators.
Main Methods:
- Enzyme inactivation assays with varying concentrations and incubation times.
- Kinetic analysis of inactivation rates.
- Radiolabeling studies to demonstrate covalent attachment of inactivators.
Main Results:
- Beta,beta-dichloro- and beta,beta,beta-trifluoroalanine act as suicide inactivators of PLP-dependent enzymes catalyzing beta- or gamma-elimination reactions.
- Inactivation is time-dependent and first-order with respect to enzyme concentration.
- Gamma-cystathionase showed stoichiometric covalent attachment of [1-14C]beta,beta,beta-trifluoroalanine.
- Monohaloalanines are substrates and do not inactivate these enzymes.
Conclusions:
- The proposed mechanism involves Schiff base formation, elimination of HCl or HF, and subsequent Michael addition of an active site nucleophile, leading to covalent enzyme modification.
- Alanine racemase is inactivated by polyhaloalanines, while glutamate-pyruvate and glutamate-oxaloacetate transaminases are inactivated by monohaloalanines but not polyhaloalanines.