Cysteine 254 can cooperate with active site cysteine 247 in reactivation of 5,5'-dithiobis(2-nitrobenzoic acid)-inactivated rhodanese as determined by site-directed mutagenesis.

D.M Miller-Martini,S Hua,P.M Horowitz

doi:10.1016/s0021-9258(18)99889-3

D.M Miller-Martini, S Hua + Show 1 more

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https://doi.org/10.1016/s0021-9258(18)99889-3

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Abstract

Sulfhydryl substitution mutants of rhodanese (thiosulfate:cyanide sulfurtransferase; EC 2.8.1.1) were used to determine whether the 4 cysteine residues in the native structure could cooperate in reactions. The sulfhydryl reactivity of persulfide-containing (ES) rhodanese was not significantly changed when cysteine residues at positions 63, 254, and 263 were replaced by serine, either individually or in combination. However, the sulfhydryl reactivity of persulfide-free (E) rhodanese was enhanced when Cys-254 was mutated. One sulfhydryl group, presumably the active site Cys-247, reacted rapidly in the E forms of these proteins with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) or 4,4'-dipyridyl disulfide (4-PDS). After reaction with DTNB or 4-PDS, proteins with Cys-254 retained > 95% of their original activities as compared with Ser-254-containing proteins, which retained < 6% of their activities. Cyanide treatment could release thionitrobenzoate from rhodanese-thionitrobenzoate complexes with an approximate 1:1 stoichiometry. After this treatment, only the wild-type and C263S enzymes were fully active. Cyanide-treated rhodanese-thionitrobenzoate complexes of the C254S and C254S/C263S mutants could be fully reactivated using an exogenously added thiol, beta-mercaptoethanol. These results are consistent with the formation of a Cys-247-thiocyano derivative that is inactive but capable of being reactivated by intramolecular transfer of cyanide to Cys-254. In the absence of Cys-254, beta-mercaptoethanol can serve as the transferring sulfhydryl group.

Highlights

~ 6 %of their activitiesC. yanide treatment could release rat liver
Suggest thaCt ys-247 is responsible for the rapidreaction in the E form. Reactions of these species with 4'-dipyridyl disulfide (4-PDS) weremore rapid than Sulfhydryl residues in rhodanese havebeen implicated in its those with Dithiobis(2-nitrobenzoic acid) (DTNB), the patterns of reactivities were folding (301, oxidative inactivation (18, 191, and catalysis(3-5, the sameas described above (Table 11;time courses not shown). 7,s)
Thereactivities of the sulfhydryl groups werneot depend- shown to be different by a variety of solution studies [21]

Summary

RESULTS

The first tyopfe fide-free (E)forms of wild-type and C254SK263Sproteins were treated reactivity wasexemplified by rhodanese species that contained to the DTNB studies except that the monitoring wavelength was 324 nmand the molar extinction coefficient of 19,800M-, cm" was used for all calculations For both the DTNB and 4-PDS reactions, the activities of the various samples, before the addition of SDS, were monitoredby dilution of 2 pl. Cyanide Releases Thionitrobenzoate from Rhodanese-Thionitrobenzoate Complexesbut Only Leads to Reactivation of Species Containing Cys-254-The E forms of the wild-type, All these species releasedTNB after treatment with cyanide as described under "Experimental Procedures." The. Time (min) release of TNB, monitored at 412 nm, vaned from approximately 0.6 moVmol of wild-type or C263S enzyme to approximately 1.5moVmo of(3254s or C254S/C263S enzyme (data not shown) and corresponded to the stoichiometry determined directly from the release of TNB on initial reaction with DTNB

This result is in keeping with the accepted mechanism for

DISCUSSION

Mer SDSb

Wild type

After the addition of immediately*