Vitamin K Oxygenation, Glutamate Carboxylation, and Processivity: Defining the Three Critical Facets of Catalysis by the Vitamin K–Dependent Carboxylase

Abstract

The vitamin K-dependent carboxylase uses vitamin K oxygenation to drive carboxylation of multiple glutamates in vitamin K-dependent proteins, rendering them active in a variety of physiologies. Multiple carboxylations of proteins are required for their activity, and the carboxylase is processive, so that premature dissociation of proteins from the carboxylase does not occur. The carboxylase is unique, with no known homology to other enzyme families, and structural determinations have not been made, rendering an understanding of catalysis elusive. Although a model explaining the relationship of oxygenation to carboxylation had been developed, until recently almost nothing was known of the function of the carboxylase itself in catalysis. In the past decade, discovery and analysis of naturally occurring carboxylase mutants has led to identification of functionally relevant residues and domains. Further, identification of nonmammalian carboxylase orthologs has provided a basis for bioinformatic analysis to identify candidates for critical functional residues. Biochemical analysis of rationally chosen carboxylase mutants has led to breakthroughs in understanding vitamin K oxygenation, glutamate carboxylation, and maintenance of processivity by the carboxylase. Protein carboxylation has also been assessed in vivo, and the intracellular environment strongly affects carboxylase function. The carboxylase is an integral membrane protein, and topological analysis, coupled with biochemical determinations, suggests that interaction of the carboxylase with the membrane is an important facet of function. Carboxylase homologs, likely acquired by horizontal transfer, have been discovered in some bacteria, and functional analysis of these homologs has the potential to lead to the discovery of new roles of vitamin K in biology. Adv. Nutr. 3: 135-148, 2012.

Characterization of vitamin K–dependent carboxylase mutations that cause bleeding and nonbleeding disorders

“…35 Since the purification of the enzyme and cloning of the gene in 1991, 36,37 significant progress has been made in understanding how GGCX interacts with its substrates and achieves catalysis. 17 Most of our knowledge concerning GGCX's function, however, has been obtained from an in vitro activity assay using the pentapeptide FLEEL as its substrate. Thus, our understanding of the carboxylation of vitamin K-dependent proteins in their native milieu is lacking.…”

“…16 It is not clear, however, why some mutations cause bleeding disorders while others cause nonbleeding syndromes. This lack of knowledge comes in part from the fact that our current understanding of GGCX's function was obtained from in vitro experimentation under artificial conditions, 17 which has limited usefulness in understanding the clinical consequences of GGCX mutations. In an attempt to address this issue, a GGCX-deficient mouse strain was generated by gene targeting.…”

The role of osteocalcin in human glucose metabolism: marker or mediator?

“…This structure is the active site for this vitamin, which acts as an enzyme cofactor. 18 Coincident with γ-carboxylation, the active quinol form of vitamin K is oxidized and subsequently recycled by a vitamin K epoxide. 17 This recycling of vitamin K consumed during γ-carboxylation can be regarded as a pathway by which to preserve limited stores of vitamin K.…”

“…This finding presents a major paradigm shift given that all known vitamin-K-dependent proteins require the presence of γ-carboxyglutamic acid for function, including the carboxylating enzyme itself. 18 …”

New Perspectives for the Nutritional Value of Vitamin K in Human Health

“…(KH2) forms, in successive reactions catalysed by vitamin K reductases [8,22,23]. The two enzymes known to be involved in this vitamin K cycle are vitamin K epoxide reductase (VKOR) and vitamin K reductase (VKR), in a process known as the vitamin K cycle [8,22].…”