Novel Mechanism of Surface Catalysis of Protein Adduct Formation: NMR STUDIES OF THE ACETYLATION OF UBIQUITIN

Jeffrey M Macdonald,Arthur L Haas,Robert E London

doi:10.1074/jbc.m000684200

Jeffrey M Macdonald, Arthur L Haas + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m000684200

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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 A distant. These studies were performed using [1-(13)C]acetyl salicylate or [1,1'-(13)C(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.

Highlights

From the Laboratory of Structural Biology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709 and the ‡Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226
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
A series of HMQC spectra corresponding to 3-h accumulation periods was obtained for both the wt ubiquitin and the H68N mutant in the presence of 40 mM [1-13C]acetyl salicylate

Summary

NMR STUDIES OF THE ACETYLATION OF UBIQUITIN*

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 This may be the first direct demonstration of a catalytic intermediate forming on the protein surface. In a number of studies involving glycation and the formation of other adducts, histidine has been proposed to play an important role in determining the reactivity of nearby lysine residues. Ubiquitin is a attractive target for such studies because of the wealth of available structural data, the lack of an active site with its specific catalytic chemistry, and the presence of a single histi- NMR studies of the modification of ubiquitin and its H68N mutant offer a more direct approach to the analysis of the role of surface functionality in protein adduct formation

EXPERIMENTAL PROCEDURES

NMR Studies of the Acetylation of Ubiquitin

RESULTS

DISCUSSION