Energy transfer provides an arrow in the metabolism of living systems. Direct energetic coupling of chemical transformations, such that the free energy generated in one reaction is channeled to another, is the essence of energy transfer, whereas the purpose is the production of high-energy chemical intermediates. Vitamin K provides a particularly instructive example of energy transfer. A key principle at work in the vitamin K system can be termed "base strength amplification." In the base strength amplification sequence, the free energy of oxygenation of vitamin K hydroquinone (vitamin KH2) is used to transform a weak base to a strong base in order to effect proton removal from selected glutamate (Glu) residues in the blood-clotting proteins.

Vitamin K is the blood-clotting vitamin. The mechanism of action of vitamin K is discussed in terms of a new carbanion model that mimics the proton abstraction from the gamma position of protein-bound glutamate. This is the essential step leading to carboxylation and activation of the blood-clotting proteins. The model comprises an oxygenation that is coupled to carbon-carbon bond formation, as is the oxygenation of vitamin K hydroquinone to vitamin K oxide. The model hypothesis is also supported by the mechanism of inhibition of the carboxylase by HCN, which acts as an acid-base inhibitor rather than a metal-complexing inhibitor. The new model postulates a dioxetane intermediate that explains the presence of a second atom of 18O (from 18O2) incorporated into vitamin K oxide in the course of the enzymatic carboxylation. Finally, the chemistry developed here has been used to define the active site of vitamin K hydroquinone as the carbon-carbon bond adjacent to the methyl group.

Vitamin K in its hydroquinone form, vitamin KH2, is a cofactor for the enzyme that carboxylates the N-terminal glutamates in six proteins of the blood-clotting cascade. Vitamin KH2 is transformed to vitamin K oxide concurrently with the carboxylation leading to -carboxyglutamate. When vitamin KH2 is treated with 1802 in the presence of rat liver microsomes, the product, vitamin K oxide, carries a full atom of 180 at the epoxide oxygen. This paper reports the partial incorporation of almost 20% of a second atom of lsO at a carbonyl oxygen of vitamin K oxide. Control reactions demonstrate that exchange with H2180 under the reaction conditions is too slow to account for the additional increment of 180. It is concluded that the second ,80 arises directly from molecular oxygen and that its incorporation is an integral part of the mechanism of action of vitamin K.

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We have discovered a novel, spontaneous model oxidation that leads to a powerful base capable of carrying out a carbon-carbon bond forming condensation reaction analogous to that observed in the vitamin K-mediated carboxylation of glutamate. The model sequence incorporates a novel base-strength enhancement sequence and it implicates molecular oxygen as the initiating factor in the vitamin K-dependent carboxylation. When the oxygenation is carried out under an atmosphere of (18)O2, two atoms of (18)O are incorporated into the vitamin K oxide product. Selective (18)O labelling defines the carbonyl group next to methyl as the active site of the vitamin K.