Publications by authors named "K Breddam"

Rare high-He/He signatures in ocean island basalts (OIB) erupted at volcanic hotspots derive from deep-seated domains preserved in Earth's interior. Only high-He/He OIB exhibit anomalous W-an isotopic signature inherited during the earliest history of Earth-supporting an ancient origin of high He/He. However, it is not understood why some OIB host anomalous W while others do not.

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In the present study we describe a novel method for obtaining highly pure carboxypeptidase Y, or derivatives thereof, in a single-step purification procedure. The method is based on affinity chromatography and the results demonstrate that an efficient method is obtained only when the affinity gel is fully saturated with enzyme. Thus, pilot experiments are required to determine the binding capacity of the resin with respect to a given enzyme.

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The S'1 binding pocket of carboxypeptidase Y is hydrophobic, spacious, and open to solvent, and the enzyme exhibits a preference for hydrophobic P'1 amino acid residues. Leu272 and Ser297, situated at the rim of the pocket, and Leu267, slightly further away, have been substituted by site-directed mutagenesis. The mutant enzymes have been characterized kinetically with respect to their P'1 substrate preferences using the substrate series FA-Ala-Xaa-OH (Xaa = Leu, Glu, Lys, or Arg) and FA-Phe-Xaa-OH (Xaa = Ala, Val, or Leu).

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The high activity of carboxypeptidase S1 with substrates having basic P1 residues is predicted to depend on the size of residue 312 in combination with the presence of a counter-charge in an alpha-helix above the S1 binding pocket. This hypothesis is tested by the construction of 32 mutant forms of carboxypeptidase Y that combines a reduction in size of residue 312 and the introduction of either a basic or an acidic residue at either position 241 or position 245. Kinetic characterization using substrates with Leu, Arg, Lys, Glu, or Asp in P1 demonstrates that most of these enzymes exhibit drastically altered catalytic properties.

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