Functional implications resulting from disruption of the calcium-binding loop in bovine alpha-lactalbumin.
The strong calcium-binding site of alpha-lactalbumin comprises the carboxylate side chains of aspartic acid 82, 87, and 88 and the carbonyl oxygens of residues 79 and 84. A single methionine residue was selectively modified by controlled CNBr cleavage to yield homoserine at position 90. The CNBr-cleaved alpha-lactalbumin lost the ability to bind calcium strongly as monitored by intrinsic fluorescence, electrophoretic mobility, atomic absorption, and x-ray fluorescence. Remarkably, the modified protein was still competent in lactose biosynthesis, although activity was reduced to 1/40th that of the native form of the protein. Although the strong calcium-binding site was destroyed as a result of the cleavage of the calcium-binding loop, a secondary calcium site was retained that directly affects a rate enhancement of lactose biosynthesis when saturated, resulting in approximately a two- to threefold increase in rate at 1 mM CaCl2 with an activation equilibrium constant of 350 +/- 40 microM.
Department of Chemistry, Ohio State University, Columbus 43210.
L J
LJ
Berliner
D C
DC
Meinholtz
Y
Y
Hirai
G
G
Musci
M P
MP
Thompson
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D007768:Lactalbumin / Q000737:chemistry* / Q000378:metabolism; D007785:Lactose / Q000096:biosynthesis; D007788:Lactose Synthase / Q000378:metabolism; D008969:Molecular Sequence Data; D013050:Spectrometry, Fluorescence
D000596:Amino Acids; D007768:Lactalbumin; D007788:Lactose Synthase; D007785:Lactose; D002118:Calcium