Novel Mechanism of Surface Catalysis of Protein Adduct Formation

Abstract

Reactivity of surface lysyl residues of proteins with a broad range of chemical agents has been proposed to be dependent on the catalytic microenvironment of the residue. We have investigated the acetylation of wild type ubiquitin and of the UbH68N mutant to evaluate the potential contribution of His-68 to the reactivity of Lys-6, which is about 4 Å distant. These studies were performed using [1-13 C]acetyl salicylate or [1,1- 13C 2 ]acetic anhydride, and the acetylated products were detected by two-dimensional heteronuclear multiple quantum coherence spectroscopy. The results demonstrate that His-68 makes a positive contribution to the rate of acetylation of Lys-6 by labeled aspirin. Additionally, a pair of transient resonances is observed after treatment of wild type ubiquitin with the labeled acetic anhydride but not upon treatment of the H68N mutant. These resonances are assigned to the acetylated His-68 residue. The loss of intensity of the acetylhistidine resonances is accompanied by an increase in intensity of the acetyl-Lys-6 peak, supporting the existence of a transacetylation process between the acetylhistidine 68 and lysine 6 residues located on the protein surface. Hence, this may be the first direct demonstration of a catalytic intermediate forming on the protein surface.

Lys6-modified Ubiquitin Inhibits Ubiquitin-dependent Protein Degradation

“…Our observation that, of the 7 Lys residues, Lys 6 is the most readily modified with sulfo-NHS-biotin in the ubiquitin molecule (Tables I and II) is consistent with prior observations that Lys 6 is the most readily modified by p-nitrophenyl acetate (28), aspirin (29), acetic anhydride (30), and Oregon green succinimidyl ester (31). Acetylation and biotinylation of Lys residues share the same mechanism as other nonenzymatic modifications such as nonenzymatic glycosylation (glycation).…”

“…This preference for mod-ification could be due to the differential amine surface accessibility (29) or to the catalytic microenvironment. The latter is most likely with respect to Lys 6 because acetylation of Lys 6 depends upon its interaction with His 68 (30). In the threedimensional structure of ubiquitin, His 68 is located in the vicinity of Lys 6 (32,33).…”

“…In the threedimensional structure of ubiquitin, His 68 is located in the vicinity of Lys 6 (32,33). The interaction between Lys 6 and His 68 provides a catalytic microenvironment for the modification of Lys 6 (30). Given the similarity between biotinylation and the chemical modifications mentioned above, Lys 6 could also be the dominant site of biotinylation.…”

“…Modifications of some Lys residues may also alter the surface property of ubiquitin chains and may interfere with the interaction between ubiquitin conjugates and the proteasome. Previous studies indicated that Lys 6 is the most susceptible residue to modification by various chemicals, such as p-nitrophenyl acetate (28), aspirin (29), acetic anhydride (30), and Oregon green succinimidyl ester (31). This preference for mod-ification could be due to the differential amine surface accessibility (29) or to the catalytic microenvironment.…”

Exogenous ubiquitin modulates chronic β-adrenergic receptor-stimulated myocardial remodeling: role in Akt activity and matrix metalloproteinase expression

“…The reasons may include differential modification of UB from different sources. UB is shown to be modified by acetylation of lysines, oxidation of methionine, and nitration of tyrosine (25,54). These modifications may influence interaction of extracellular UB with its receptor and/or other proteins.…”