The local electrostatic environment determines cysteine reactivity of tubulin |
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Authors: | Britto P J Knipling Leslie Wolff J |
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Institution: | Laboratory of Biochemistry and Genetics, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA. |
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Abstract: | Of the 20 cysteines of rat brain tubulin, some react rapidly with sulfhydryl reagents, and some react slowly. The fast reacting cysteines cannot be distinguished with 14C]iodoacetamide, N-(14)C]ethylmaleimide, or IAEDANS (5-((((2-iodoacetyl)amino)ethyl)amino) naphthalene-1-sulfonic acid]), since modification to mole ratios 1 cysteine/dimer always leads to labeling of 6-7 cysteine residues. These have been identified as Cys-305alpha, Cys-315alpha, Cys-316alpha, Cys-347alpha, Cys-376alpha, Cys-241beta, and Cys-356beta by mass spectroscopy and sequencing. This lack of specificity can be ascribed to reagents that are too reactive; only with the relatively inactive chloroacetamide could we identify Cys-347alpha as the most reactive cysteine of tubulin. Using the 3.5-A electron diffraction structure, it could be shown that the reactive cysteines were within 6.5 A of positively charged arginines and lysines or the positive edges of aromatic rings, presumably promoting dissociation of the thiol to the thiolate anion. By the same reasoning the inactivity of a number of less reactive cysteines could be ascribed to inhibition of modification by negatively charged local environments, even with some surface-exposed cysteines. We conclude that the local electrostatic environment of cysteine is an important, although not necessarily the only, determinant of its reactivity. |
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