Purification to apparent homogeneity and properties of pig kidney L-fucose kinase.
L-Fucokinase was purified to apparent homogeneity from pig kidney cytosol. The molecular mass of the enzyme on a gel filtration column was 440 kDa, whereas on SDS gels a single protein band of 110 kDa was observed. This 110-kDa protein was labeled in a concentration-dependent manner by azido-[32P]ATP, and labeling was inhibited by cold ATP. The 110-kDa protein was subjected to endo-Lys-C digestion, and several peptides were sequenced. These showed very little similarity to other known protein sequences. The enzyme phosphorylated L-fucose using ATP to form beta-L-fucose-1-P. Of many sugars tested, the only other sugar phosphorylated by the purified enzyme was D-arabinose, at about 10% the rate of L-fucose. Many of the properties of the enzyme were determined and are described in this paper. This enzyme is part of a salvage pathway for reutilization of L-fucose and is also a valuable biochemical tool to prepare activated L-fucose derivatives for fucosylation reactions.
Park
S H
SH
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA.
Pastuszak
I
I
Drake
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Elbein
A D
AD
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000345:Affinity Labels / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001089:Arabinose / Q000378:metabolism; D002236:Carbohydrate Conformation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005643:Fucose / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism; D006600:Hexosephosphates / Q000032:analysis / Q000378:metabolism; D007668:Kidney / Q000201:enzymology*; D008666:Metalloendopeptidases / Q000378:metabolism; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000302:isolation & purification* / Q000378:metabolism; D017421:Sequence Analysis; D013379:Substrate Specificity; D013552:Swine
D000345:Affinity Labels; D004791:Enzyme Inhibitors; D006600:Hexosephosphates; D008670:Metals; C081214:fucopyranosyl phosphate; D005643:Fucose; D000255:Adenosine Triphosphate; D001089:Arabinose; D017853:Phosphotransferases (Alcohol Group Acceptor); C020153:fucokinase; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase