Mutants double, histidine

The pathway for proton transfer to QB is studied in the reaction center (RC) from Rb. sphaeroides using two approaches (Adelroth et al., 2001) 1) the binding of Zn2+ or Cd2+ to the RC surface at His-H126, His-H128, and Asp-H124 and 2) the replacement of the histidines for Ala. In the double mutant RC at pH 8.5, the observed rates of proton uptake associated with both the first and the second proton-coupled electron-transfer... 

One recombinant FetSp mutant is unique among multicopper oxidase species and has been particularly informative about the structure of the type 3 binuclear cluster in these species. This is the T1D/T2D double mutant that contains only this type 3 site (Blackburn et al., 2000). EXAFS analysis of this protein contains contributions from electron ejection and scattering from only the type 3 copper atoms and thus provides direct structural information about this cluster. The K-edge XAS spectrum for this mutant in its oxidized and reduced states is shown in Fig. 21. The oxidized sample has a nearly featureless edge with a midpoint energy of 8990 eV typical of tetragonally distorted type 2 Cu(ll) centers, i.e., those with predominantly histidine imidazole coordination. The reduced type 3 cluster exhibited a pronounced shoulder at 8984 eV just below the... 

A double mutant of BuChE containing both histidine (rather than glycine) at amino acid residue 117 and glutamine in place... 

As expected for a biosynthetic pathway [7], the activity of the first enzyme (PR-ATP synthetase ) is controlled by the end product of the pathway [8]. At the pH used in the standard assay, pH 8.5, feedback inhibition by histidine is noncompetitive with respect to both substrates (PRPP and ATP), having a Ki of 100 fiM. When the pH is reduced to one more likely representative of that in the cell, pH 7.5, the strength of the inhibition is about doubled [8]. Feedback-resistant mutants of the PR-ATP s)mthetase have been obtained by selecting for resistance to the histidine analog 2-thiazolealanine (for structure, see Fig. 2). This analog does not substitute for histidine in proteins, but inhibits the PR-ATP synthetase [8,11,12], and thereby produces bacteriostasis in the sensitive cell by cutting off its histidine supply. Feedback-resistant mutants excrete Itistidine into the medium, whereas... 

HisU mutants have many properties in common with hisW mutants, suggesting that their mutations pertain to related functions. These functions cannot be identical, however, since a double dose of a hisW gene does not allow repression in a hisU strain [109]. Like hisW mutants, hisU mutants have about a 25% reduction in their level of histidine tRNA when compared to the acceptance of several other tRNA s, and about a 45 % reduction when compared to the total amount of nucleic acid extracted [108]. In addition, both mutant types are resistant to the proline analog, L-azetidine-2-carboxylic acid [113], and the derepression level of both responds in the same manner to different growth media (see Section IV, K). These similarities, and in particular those concerned with the level of histidine tRNA in the mutants, suggest that hisU is also involved with tRNA maturation. 

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