Sulfhydryl groups of pyocin R1. Morphology and activity modified with sulfhydryl reagents.

Electron microscopic observation of pyocin R1 with the negative staining technique demonstrated that pyocin R1 retained its phage tail-like shape of an extended sheath even when it was inactivated by treatment with p-chloromercuribenzoic acid (PCMB) or 4-(p-sulfophenylazo)-2-mercuriphenol (SAMP). Thus it was shown that the contraction and extension of the sheath does not occur reversibly on the modification of sulfhydryl groups accompanying the change of activity. The activity lost under these conditions was restored to the original level by treatment with 2-mercaptoethanol (2-ME). Numbers of sulfhydryl groups in the pyocin R1 particle were determined to be 208 mol and 152 mol per mol (11.8 x 10(6) daltons) by spectrophotometric titration with SAMP and by membrane-filter assay with radioactive PCMB, respectively. Most of these cysteine residues appeared to be localized in the substructure other than the sheath and core. It was also shown that all of these sulfhydryl groups were not necessary for expression of its activity but a part of them were essential for adsorption to the sensitive cells.     

Study of reactivity of sulfhydryl groups of troponin]

The reactivities of SH-groups of troponin and its components were studied, using 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). At low concentrations of Ca2+ (pCa greater than 8) one rapidly- and two slowly-reacting SH-groups of troponin I and one SH-group of troponin C slowly reacting with NBD-chloride are titrated in the whole troponin complex. When Ca2+ concentration is increased up to pCa less than 5, only one slowly-reacting and one rapidly-reacting with NBD-chloride SH-groups of troponin I are titrated. The increase of Ca2+ concentration from pCa greater than 8 to pCa less than 5 results in a change of the environment polarity for the highly reactive SH-group of troponin I in the whole troponin complex. This phenomenon may suggest that the changes in troponin C structure during Ca2+ binding somehow induce changes in that of troponin I. The half-maximal change of troponin SH-groups reactivity is found at pCa 6.8. No cooperativity for Ca2+ binding by the troponin complex is observed using SH-groups titration by NBD-Cl.     

Physiological activity of warburganal and its reactivity with sulfhydryl groups

Warburganal, a unique dialdehyde sesquiterpene isolated from East African Warburgia plants, showed a strong antifungal activity. However, this growth inhibition in Saccharomyces cerevisiae was reversed with L-cysteine. In addition, warburganal inhibited the alcoholic fermentation of S. cerevisiae while L-cysteine reversed this inhibition. When alcohol dehydrogenase, a sulfhydryl enzyme, was incubated with warburganal, the enzyme activity decreased with time. The decrease was more rapid at alkaline pH. L-Cysteine prevented this enzyme inhibition by warburganal but could not restore the enzyme activity lost already due to warburganal. Warburganal lost its characteristic ultraviolet absorption spectrum in the presence of L-cysteine. The change in absorbance was favored at alkaline pH, indicating Michael reaction type addition of L-cysteine to warburganal. Based on these observations, a variety of physiological activities due to warburganal appear to result from its irreversible reactivity with sulfhydryl groups.     

Function and reactivity of sulfhydryl groups of rat liver glycine methyltransferase

Rat liver glycine methyltransferase is completely inactivated by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Treatment of the inactivated enzyme with KCN results in a reactivated enzyme having values of Vmax and S0.5 for S-adenosyl-L-methionine comparable to those of the native enzyme and about a 4-fold greater Km value for glycine. Kinetics of inactivation and reactivation show that one cysteine residue is involved in this process. Reaction of the methyltransferase with iodoacetate leads to partial inactivation of the enzyme; about 22% of the initial activity is retained in the modified enzyme. The relationship between the loss of enzyme activity and the number of iodoacetate molecules incorporated and the sequence analysis of peptides containing the modified residues indicate that carboxymethylation of Cys-282 is responsible for loss of activity. The observations that the activity of the cyanylated glycine methyltransferase shows no decrease upon incubation with iodoacetate and, conversely, the residual activity associated with the iodoacetate-modified enzyme is not abolished by DTNB suggest that Cys-282 is also involved in the inactivation by DTNB. Besides this residue, Cys-185, Cys-246, and Cys-262 are modified upon prolonged incubation with iodoacetate. 5'-[p-(Fluorosulfonyl)benzoyl]adenosine (FSBA) inactivates glycine methyltransferase by forming 1 disulfide/subunit [Fujioka, M., & Ishiguro, Y. (1986) J. Biol. Chem. 261, 6346-6351]. Despite this stoichiometry, treatment of the FSBA-inactivated enzyme with unlabeled iodoacetate and then with iodo[14C]acetate after reduction with 2-mercaptoethanol and subsequent peptide analysis show that the incorporated radioactivity is distributed equally among Cys-185, Cys-246, Cys-262, and Cys-282.(ABSTRACT TRUNCATED AT 250 WORDS)     

The reactivity of sulfhydryl groups of bovine cardiac troponin C

Bovine cardiac troponin C (cTnC) contains 2 cysteine residues, Cys-35 located in the nonfunctional Ca2+-binding loop I and Cys-84 in the N-terminal segment of the central helix. We have studied the reactivity of Cys residues in cTnC with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM). The latter compound fluoresces only when reacted with the protein. The reaction with DTNB followed second order kinetics with respect to DTNB, the rate constants being 3.37 s-1 M-1 and 1.82 s-1 M-1 in the presence and absence of Ca2+, respectively. These rates are much slower than the rate of reaction with Cys-98 of skeletal TnC (sTnC) or with the urea-denatured cTnC, indicating that both Cys residues are partly buried within the structure of the protein. The increase in reactivity was induced by binding of Ca2+ to the single low affinity Ca2+ binding site (site II). The fluorescence increase upon reaction of cTnC with CPM in the absence of Ca2+ could be fitted with a single exponential equation indicating that both cysteine residues are equally available to the reagent. The reaction in the presence of Ca2+ was biphasic. Analysis of CNBr fragments of cTnC labeled with CPM under various conditions indicated that in the presence of Ca2+ the reactivity of Cys-84 is increased while that of Cys-35 is slightly decreased. This finding is consistent with the model of Herzberg et al. (Herzberg, O., Moult, J., and James, M. N. G. (1986) J. Biol. Chem. 261, 2638-2644) and the data of Ingraham and Hodges (Ingraham, R. H., and Hodges, R. S. (1988) Biochemistry 27, 5891-5898), suggesting that the Ca2+-induced conformational change in the N-terminal half of TnC involves separation of the helix C from the central helix, thereby increasing the accessibility of Cys-84. The slow overall kinetics, however, indicates that the structure in the vicinity of Cys residues is relatively compact regardless of Ca2+. We interpret the increase in reactivity towards CPM as consistent with a Ca2+-induced exposure of a hydrophobic pocket in the vicinity of Cys-84.     

Recombinant rat liver guanidinoacetate methyltransferase: reactivity and function of sulfhydryl groups

Rat liver guanidinoacetate methyltransferase, produced in Escherichia coli by recombinant DNA technique, possesses five cysteine residues per molecule. No disulfide bond is present. Analysis of the chymotryptic peptides derived from the iodo[14C]acetate-modified enzyme shows that Cys-90, Cys-15, Cys-219, and Cys-207 are alkylated by the reagent in order of decreasing reactivity. Incubation of the enzyme with excess 5,5'-dithiobis(2-nitrobenzoate) (DTNB) in the absence and presence of cystamine [2,2'-dithiobis(ethylamine)] causes the appearance of 4 and 5 mol of 2-nitro-5-mercaptobenzoate/mol of enzyme, respectively. Reaction of the methyltransferase with an equimolar amount of DTNB results in an almost quantitative disulfide cross-linking of Cys-15 and Cys-90 with loss of a large portion of the activity. The methyltransferase is completely inactivated by iodoacetate following nonlinear kinetics. Comparison of the extent of inactivation with that of modification of cysteine residues and the experiment with the enzyme whose Cys-15 and Cys-90 are cross-linked suggest that alkylation of Cys-15 and Cys-90 results in a partially active enzyme and that carboxymethylation of Cys-219 completely eliminates enzyme activity. The inactivation of guanidinoacetate methyltransferase by iodoacetate or DTNB is not protected by substrates. Furthermore, disulfide cross-linking of Cys-15 and Cys-90 or carboxymethylation of Cys-219 does not impair the enzyme's capacity to bind S-adenosylmethionine. Thus, these cysteine residues appear to occur outside the active-site region, but their integrity is crucial for the expression of enzyme activity.     

The reactivity of sulfhydryl groups of yeast DNA dependent RNA polymerase I

The number of reactive cysteine residues of yeast RNA polymerase I was determined and their function was studied using parachloromercury benzoate (pCMB), dithiobisnitrobenzoate (DTNB) and N-ethyl-maleimide (NEM) as modifying agents. By treatment with DTNB about 45 sulfhydryl groups react in the presence of 8M urea. Under non-denaturing conditions only 20 sulfhydryl groups are reactive with pCMB and DTNB. Both reagents completely inactivate the enzyme and this effect can be reversed by reducing agents. The sedimentation coefficient and the subunit composition are not affected when the enzyme is inactivated. Two of the most reactive sulfhydryl groups are necessary for activity. The modification of these groups is partially protected by substrates and DNA, suggesting that they are involved either in catalysis or in the maintenance of the conformation of the active site. Experiments with 14C-NEM indicate that the most reactive groups are located in subunits of 185,000, 137,000 and 41,000 daltons.     

Histidine decarboxylase of Lactobacillus 30a: function and reactivity of sulfhydryl groups.

Two classes of sulfhydryl groups in histidine decarboxylase from Lactobacillus 30 a can be differentiated by their reaction with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Five cysteinyl residues (class I) of the native enzyme are titrated by DTNB as the pH of the reaction medium is increased from 6.5 to 7.5; the pH-rate profile for their reaction is described by a pKa of 9.2. An additional five thiol groups (class II) are titrated only when denaturing agents are added above neutral pH. Histidine decarboxylase is completely inactivated by DTNB in a kinetically second-order process (Kapp = 660 +/- 20 M-1 min-1 at pH 7.6 and 25 degrees C) which occurs coincident with and at the same rate as modification of the five class-I SH groups of the enzyme, i.e., one thiol group per pyruvoyl prosthetic group. The competitive inhibitors, histamine and imidazole, markedly enhanced the reactivity of these cysteinyl residues toward DTNB; this enhancement is accompanied by a concomitant increase in the rate of inactivation. A single SH group in each of the five catalytic units of histidine decarboxylase is thus implicated as being critical for the expression of enzymatic activity.