title: The local electrostatic environment determines cysteine reactivity of tubulin.
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.
authors.affiliation: Laboratory of Biochemistry and Genetics, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: P J
authors.initials: PJ
authors.lastname: Britto
authors.forename: Leslie
authors.initials: L
authors.lastname: Knipling
authors.forename: J
authors.initials: J
authors.lastname: Wolff
mesh_terms: D000081:Acetamides / Q000737:chemistry; D000818:Animals; D003545:Cysteine / Q000378:metabolism*; D004583:Electrons; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007460:Iodoacetamide / Q000494:pharmacology; D007700:Kinetics; D008024:Ligands; D008239:Lysine / Q000737:chemistry; D013058:Mass Spectrometry; D008958:Models, Molecular; D010169:Palmitic Acids / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D051381:Rats; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D055672:Static Electricity; D013440:Sulfides; D013997:Time Factors; D014357:Trypsin / Q000378:metabolism; D014404:Tubulin / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000081:Acetamides; D004791:Enzyme Inhibitors; D008024:Ligands; D010169:Palmitic Acids; D010455:Peptides; D013440:Sulfides; D014404:Tubulin; C013874:chloroacetamide; D006639:Histidine; D014357:Trypsin; D008239:Lysine; D003545:Cysteine; D007460:Iodoacetamide