Expression and characterization of the naturally occurring mutation L394R in human gamma-glutamyl carboxylase.

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Mutucumarana VP, Stafford DW, Stanley TB, Jin DY, Solera J, Brenner B, Azerad R, Wu SM

Expression and characterization of the naturally occurring mutation L394R in human gamma-glutamyl carboxylase.

J Biol Chem. 2000 Oct 20;275(42):32572-7.

PubMed ID
10934213 [ View in PubMed
]
Abstract

Patients with mutation L394R in gamma-glutamyl carboxylase have a severe bleeding disorder because of decreased biological activities of all vitamin K-dependent coagulation proteins. Vitamin K administration partially corrects this deficiency. To characterize L394R, we purified recombinant mutant L394R and wild-type carboxylase expressed in baculovirus-infected insect cells. By kinetic studies, we analyzed the catalytic activity of mutant L394R and its binding to factor IX's propeptide and vitamin KH(2). Mutant L394R differs from its wild-type counterpart as follows: 1) 110-fold higher K(i) for Boc-mEEV, an active site-specific, competitive inhibitor of FLEEL; 2) 30-fold lower V(max)/K(m) toward the substrate FLEEL in the presence of the propeptide; 3) severely reduced activity toward FLEEL carboxylation in the absence of the propeptide; 4) 7-fold decreased affinity for the propeptide; 5) 9-fold higher K(m) for FIXproGla, a substrate containing the propeptide and the Gla domain of human factor IX; and 6) 5-fold higher K(m) for vitamin KH(2). The primary defect in mutant L394R appears to be in its glutamate-binding site. To a lesser degree, the propeptide and KH(2) binding properties are altered in the L394R mutant. Compared with its wild-type counterpart, the L394R mutant shows an augmented activation of FLEEL carboxylation by the propeptide.

DrugBank Data that Cites this Article

Polypeptides
NameUniProt ID
Vitamin K-dependent gamma-carboxylaseP38435Details