Functional Implications Resulting from Disruption of the Calcium-Binding Loop in Bovine -Lactalbumin

¹ The Department of Chemistry, The Ohio State University, 120 West 18th Avenue, Columbus 43210
² The Department of Biochemistry, Nippon Medical School, Tokyo, Japan
³ University of Rome "La Sapienza", Department of Biochemical Sciences, Piazzale Aldo Mora 5, Rome, Italy
⁴ US Department of Agriculture, Eastern Regional Research Center, Philadelphia, PA 19118

The strong calcium-binding site of -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 -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 1/4Oth 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 1mM CaCl₂ with an activation equilibrium constant of 350 ± 40 µM.

Key Words: calcium-binding loop disruption • functionality • -lactalbumin

Submitted on September 19, 1990
Accepted on November 5, 1990