Effect of vitamin K-dependent protein precursor propeptide, vitamin K hydroquinone, and glutamate substrate binding on the structure and function of {gamma}-glutamyl carboxylase.
The γ-glutamyl carboxylase utilizes four substrates to catalyze carboxylation of certain glutamic acid residues in vitamin K-dependent proteins. How the enzyme brings the substrates together to promote catalysis is an important question in understanding the structure and function of this enzyme. The propeptide is the primary binding site of the vitamin K-dependent proteins to carboxylase. It is also an effector of carboxylase activity. We tested the hypothesis that binding of substrates causes changes to the carboxylase and in turn to the substrate-enzyme interactions. In addition we investigated how the sequences of the propeptides affected the substrate-enzyme interaction. To study these questions we employed fluorescently labeled propeptides to measure affinity for the carboxylase. We also measured the ability of several propeptides to increase carboxylase catalytic activity. Finally we determined the effect of substrates: vitamin K hydroquinone, the pentapeptide FLEEL, and NaHCO(3), on the stability of the propeptide-carboxylase complexes. We found a wide variation in the propeptide affinities for carboxylase. In contrast, the propeptides tested had similar effects on carboxylase catalytic activity. FLEEL and vitamin K hydroquinone both stabilized the propeptide-carboxylase complex. The two together had a greater effect than either alone. We conclude that the effect of propeptide and substrates on carboxylase controls the order of substrate binding in such a way as to ensure efficient, specific carboxylation.
Higgins-Gruber
Shannon L
SL
Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
Mutucumarana
Vasantha P
VP
Lin
Pen-Jen
PJ
Jorgenson
James W
JW
Stafford
Darrel W
DW
Straight
David L
DL
D000818:Animals; D019735:Carbon-Carbon Ligases / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D051379:Mice; D009842:Oligopeptides / Q000737:chemistry* / Q000378:metabolism; D011498:Protein Precursors / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D023721:Tetraodontiformes; D024482:Vitamin K 2 / Q000737:chemistry* / Q000378:metabolism
D009842:Oligopeptides; D011498:Protein Precursors; D024482:Vitamin K 2; D019735:Carbon-Carbon Ligases; C020660:glutamyl carboxylase