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Thiol-disulphide interchange in tubulin: kinetics and the effect on polymerization.

ABSTRACT: All 20 cysteine residues are accessible to disulphide reagents in the tubulin dimer, whereas only four are accessible in taxol-stabilized microtubules. Reaction rates with disulphide reagents are a function of the reagent, are decreased by G nucleotides, and increased with increase in pH and urea. With transient (stop-flow) kinetics, DTNB [5,5'-dithiobis-(2-nitrobenzoic acid)] and 2,2'-dithiodipyridine progress curves cannot be fitted by the sum of exponential terms based only on classes of cysteines. The mixed disulphide products react further to form both intra- and intermonomer disulphide bonds that can be reversed by reducing agents. With MMTS (methyl methanethiosulphonate) or ODNB (n-octyl-dithio-2-nitrobenzoate), virtually no protein-protein disulphide bonds are formed and the ODNB reaction can be given as the sum of three exponential terms with pseudo-first-order rate constants of 0.206, 0.069 and 0.010 s(-1) at pH 6.5, suggesting three classes of thiol reactivities. Limited cysteine substitution leads to only small changes in tryptophan or CD spectra, whereas complete substitution leads to loss of the helix content. MMTS-induced loss of SH groups leads to progressive increases in the critical concentration and loss of polymerization competence that can be reversed by assembly promoters such as higher protein concentration, taxol or high ionic strength. Under such conditions, the substituted tubulin forms protofilament-based structures such as microtubules, open tubules, sheets and/or bundles.

SUBMITTER: Britto PJ

PROVIDER: S-EPMC1175133 | biostudies-other | 2005 Jul

REPOSITORIES: biostudies-other

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Project description:The protein disulphide-bond isomerization activity of highly active homogeneous protein disulphide-isomerase (measured by re-activation of 'scrambled' ribonuclease) is enhanced by EDTA and by phosphate buffers. As shown for previous less-active preparations, the enzyme has a narrow pH optimum around pH 7.8 and requires the presence of either a dithiol or a thiol. The dithiol dithiothreitol is effective at concentrations 100-fold lower than the monothiols reduced glutathione and cysteamine. The enzyme follows Michaelis-Menten kinetics with respect to these substrates; Km values are 4,620 and 380 microM respectively. The enzyme shows apparent inhibition by high concentrations of thiol or dithiol compounds (greater than 10 X Km), but the effect is mainly on the extent of reaction, not the initial rate. This is interpreted as indicating the formation of significant amounts of reduced ribonuclease in these more reducing conditions. The purified enzyme will also catalyse net reduction of insulin disulphide bonds by reduced glutathione (i.e. it has thiol:protein-disulphide oxidoreductase or glutathione:insulin transhydrogenase activity), but this requires considerably higher concentrations of enzyme and reduced glutathione than does the disulphide-isomerization activity. The Km for reduced glutathione in this reaction is an order of magnitude greater than that for the disulphide-isomerization activity, and the turnover number is considerably lower than that of other enzymes that can catalyse thiol-disulphide oxidoreduction. Conventional two-substrate steady-state analysis of the thiol:protein-disulphide oxidoreductase activity indicates that it follows a ternary-complex mechanism. The protein disulphide-isomerase and thiol:protein-disulphide oxidoreductase activities co-purify quantitatively through the final stages of purification, implying that a single protein species is responsible for both activities. It is concluded that previous preparations, from various sources, that have been referred to as protein disulphide-isomerase, disulphide-interchange enzyme, thiol:protein-disulphide oxidoreductase or glutathione:insulin transhydrogenase are identical or homologous proteins. The assay, nomenclature and physiological role of this enzyme are discussed.

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Thiol-disulphide interchange in tubulin: kinetics and the effect on polymerization.

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