An approach to trapping .gamma.-glutamyl radical intermediates proposed for vitamin K dependent carboxylase: .alpha.,.beta.-methyleneglutamic acid

Abstract

The vitamin K dependent carboxylase activates the glutamyl gamma-CH of substrate peptides for carboxylation by producing a gamma-glutamyl free radical, a gamma-glutamyl carbanion, or through a concerted carboxylation. We propose to intercept the putative gamma-glutamyl free radical by the intramolecular rearrangement of a substrate containing the alpha,beta-cyclopropane analogue of glutamic acid. The rearrangement of cyclopropylcarbinyl radicals into 2-butenyl radicals is rapid, exothermic, and considered diagnostic of free-radical formation. 1-Amino-2-(carboxymethyl)cyclopropane-1-carboxylate, the beta-cyclopropane analogue of glutamic acid, was synthesized starting from diethyl alpha-ketoglutarate. The alpha-keto ester was first treated with benzonitrile in sulfuric acid, to yield diethyl alpha,alpha-dibenzamidoglutarate. The alpha,alpha-dibenzamido acid was cleaved to produce the alpha,beta-dehydroamino acid and benzamide on treatment with p-toluenesulfonic acid in hot benzene. Diazomethane addition to the dehydroamino acid resulted in cycloaddition of diazomethane and production of the pyrazoline, which upon irradiation lost N2 to give the protected cyclopropane-containing amino acid analogue. Acidic hydrolysis of the N-benzoyl-alpha,beta-methyleneglutamate diethyl ester resulted in the production of the unprotected amino acid, alpha,beta-methyleneglutamic acid, in high yield. A single dehydroamino acid and a single methyleneglutamic acid isomer were produced in this synthesis; both are identified as the Z isomer, the former by NMR using the nuclear Overhauser effect and the latter through X-ray crystallographic analysis of N-benzoyl-alpha,beta-methyleneglutamate diethyl ester. Saponification of a N-protected methyleneglutamic acid dialkyl ester using limiting alkali was shown to selectively yield the alpha-alkyl ester gamma-acid. The reaction was used to produce alpha,beta-cyclopropane-containing analogues of the carboxylase substrates N-t-Boc-L-glutamic acid alpha-benzyl ester and N-benzoyl-L-glutamic acid alpha-ethyl ester. The cyclpropane-containing analogues were tested and found to be neither substrates for nor inhibitors of the rat liver microsomal vitamin K dependent carboxylase. The inability of the enzyme to recognize these substrate analogues is attributed to the alpha-alkyl substitution, which apparently abolishes substrate binding.

Vitamin K and Energy Transduction: A Base Strength Amplification Mechanism

“…Two mechanistic possibilities, a free-radical path (14)(15)(16), and one involving a carbanion intermediate (17), have been considered. Thermochemical arguments show (13), as do experimental studies, that free-radical carboxylations are thermochemically unfavorable by 10 highly favorable in thermochemical terms.…”

Propeptide and glutamate-containing substrates bound to the vitamin K-dependent carboxylase convert its vitamin K epoxidase function from an inactive to an active state

“…[6][7][8]. Mechanisms involving either free radical abstraction of a ␥-hydrogen (12)(13)(14) or formation of a carbanion at the ␥-carbon (15,16) have been proposed. Oxygen and vitamin KH 2 -dependent exchange of tritium from tritiated water to the ␥-carbon of a glutamic acid in a carboxylase substrate at low CO 2 concentrations can be taken as evidence of a carbanion mechanism (17).…”

Mechanism of cyanide inhibition of the blood-clotting, vitamin K-dependent carboxylase.

“…This article must therefore be hereby marked "advertisement" in accordance with 18 U.S. 25) under normal biochemical circumstances has encouraged exploration of alternative hypotheses, such as that involving free radicals (18).…”