Galactose oxidase is a member of a growing class of proteins with novel posttranslationally modified redox-active amino acids (see Figure 1). 1 The unusual nature of these modifications has stimulated interest in the mechanisms by which such cofactors are generated. Recently, the biogenesis of the 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor of amine oxidase has been defined. 2 The oxidation of tyrosine to TPQ requires only copper ions and dioxygen, and is not dependent on any accessory proteins. 3 Analogous experiments with galactose oxidase have been hampered by the lack of sufficient quantities of pure precursor (unprocessed, copper-free) protein. Here we report the isolation of an apo, pro-enzyme form of galactose oxidase, and demonstrate that cleavage of the pro-sequence and assembly of the characteristic Tyr • -Cys cofactor are self-processing reactions. Figure 1 illustrates the critical features of the galactose oxidase active site. 4 In the oxidized (active) state tyrosine 272, which is posttranslationally cross-linked to cysteine 228 via a thioether bond, is oxidized to a radical. Thus the [Cu(II) Tyr • -Cys] unit acts as a two-electron acceptor in the oxidation of a wide variety of alcohols to the corresponding aldehydes. The posttranslational cross-link is believed to modulate the reactivity and redox potential of the tyrosyl radical. 5,6 C228 may aid in stabilization of the radical by virtue of the electron-donating properties of the sulfur atom. 5 The oxidized form of galactose oxidase displays a characteristic set of electronic transitions (vida infra) that are also observed in glyoxal oxidase, a galactose oxidase homologue. 7 Heterologous expression 8 of the Fusarium protein in Aspergillus nidulans under copper-limited conditions resulted in the appearance of multiple protein forms ( Figure 2). The molecular weights of the SDS-PAGE bands in Figure 2a, established to be galactose oxidase by Western blotting, 8 were estimated as 70.2, 68.5, and ∼65.5 kDa. N-terminal sequencing established that the fastest migrating protein (lower band, ∼65.5 kDa) corresponds to mature, wild-type galactose oxidase. Mature galactose oxidase migrates on SDS-PAGE with an anomalous molecular weight (65.5 kDa as compared to 68.5 kDa predicted by the sequence), owing to the thioether bond, which produces a stable loop thus preventing full unfolding on treatment with SDS. 8 The middle band ( Figure 2a) has an estimated M r that correlates with the mass of the mature galactose oxidase amino acid sequence, suggesting that it is a form of galactose oxidase lacking the thioether bond. This behavior is mirrored by the variant C228G, which is unable to generate a thioether bond. 8 Finally, the upper band (Figure 2a), having an estimated M r of 70.2 kDa, corresponds to the pro-form with the pro-sequence attached, which was confirmed by the N-terminal sequence data (Table 1). These data suggest that prosequence cleavage and thioether bond formation are separable reactions in vivo.Purification of a homogeneous form of u...

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Divalent and tetravalent analogues of ganglioside GM1 are potent inhibitors of cholera toxin and Escherichia coli heat-labile toxin. However, they show little increase in inherent affinity when compared to the corresponding monovalent carbohydrate ligand. Analytical ultracentrifugation and dynamic light scattering have been used to demonstrate that the multivalent inhibitors induce protein aggregation and the formation of space-filling networks. This aggregation process appears to arise when using ligands that do not match the valency of the protein receptor. While it is generally accepted that multivalency is an effective strategy for increasing the activity of inhibitors, here we show that the valency of the inhibitor also has a dramatic effect on the kinetics of aggregation and the stability of intermediate protein complexes. Structural studies employing atomic force microscopy have revealed that a divalent inhibitor induces head-to-head dimerization of the protein toxin en route to higher aggregates.

DNA packaging in the bacteriophage φ29 involves a molecular motor with protein and RNA components, including interactions between the viral connector protein and molecules of pRNA, both of which form multimeric complexes. Data are presented to demonstrate the higher order assembly of pRNA together with the affinity of pRNA:pRNA and pRNA:connector interactions, which are used to propose a model for motor function. In solution, pRNA can form dimeric and trimeric multimers in a magnesium-dependent manner, with dissociation constants for multimerization in the micromolar range. pRNA:connector binding is also facilitated by the presence of magnesium ions, with a nanomolar apparent dissociation constant for the interaction. From studies with a mutant pRNA, it appears that multimerization of pRNA is not essential for connector binding and it is likely that connector protein is involved in the stabilization of higher order RNA multimers. It is proposed that magnesium ions may promote conformational change that facilitate pRNA:connector interactions, essential for motor function.