Tyr 146 of TS has been proposed to assist in the removal of the proton from the 5-carbon of the pyrimidine in a steady-state intermediate [Hyatt, D. C., Maley, F., and Montfort, W. R. (1997) Biochemistry 36, 4585-4594]. We prepared a replacement set of mutations at position 146 of L. casei TS. The kcat and kcat/Km values of 15 mutants studied were significantly lower than wild-type TS. There was no effect on the Km of dUMP, and only moderate effects on the Km of the cofactor. We concluded that Y146 is not directly involved in substrate binding, but contributes significantly to catalysis. We also examined the Y146 mutants as catalysts for cofactor-independent dehalogenation of BrdUMP, a reaction which simulates early steps of the normal pathway up to and including enzyme-nucleotide covalent adduct formation. Many mutants had activity comparable to the wild-type enzyme, and we concluded that the effects of Tyr 146 mutations occur after the initial covalent adduct is formed. A covalent steady-state intermediate-containing enzyme, dUMP, and cofactor accumulated with Tyr 146 mutants, and could be isolated by SDS-PAGE. The complex was kinetically competent as an intermediate in dTMP formation. Using Y146D and F, it was shown that removal of the C-5 proton from the covalent intermediate was defective. We conclude that in the wild-type enzyme Tyr 146 assists in proton removal from the covalent intermediate. Mutants containing fluorinated tyrosines at position 146 showed an inverse linear correlation of activity versus acidity, again indicating that the basicity of the phenolic oxygen plays an important catalytic role. Speculations of how the poorly basic phenol group might assist proton removal are made in which Tyr 146 acts as a proton conduit to N5 of the cofactor or as a cohort of a water molecule serving as the direct general base catalyst.
Vc-NhaD is a Na؉ /H ؉ antiporter from Vibrio cholerae with a sharp maximum of activity at pH ϳ 8.0. NhaD homologues are present in many bacteria as well as in higher plants. However, very little is known about structure-function relations in NhaD-type antiporters. In this work 14 conserved polar residues associated with putative transmembrane segments of Vc-NhaD have been screened for their possible role in the ion translocation and pH regulation of Vc-NhaD. Substitutions S150A, D154G, N155A, N189A, D199A, T201A, T202A, S389A, N394G, S428A, and S431A completely abolished the Vc-NhaD-mediated Na These data suggest that side chains of His-93 and His-210 are involved in proton binding and that His-93 also contributes to the binding of Na ؉ ions during the catalytic cycle. These 15 residues are clustered in three distinct groups, two located at opposite sides of the membrane, presumably facilitating the access of substrate ions to the third group, a putative catalytic site in the middle of lipid bilayer. The distribution of these key residues in Vc-NhaD molecule also suggests that transmembrane segments IV, V, VI, X, XI, and XII are situated close to one another, creating a transmembrane relay of charged/polar residues involved in the attraction, coordination, and translocation of transported cations.Sodium proton antiporters are universal secondary ion transporters in bacteria. Typically, they expel toxic Na ϩ and Li ϩ ions from the cytoplasm at the expense of the proton motive force, thus playing an important role in cytoplasmic Na ϩ and pH homeostasis and providing energy for Na ϩ symports (for review, see Refs. 1-4 29 -30). This distinguishes Vc-NhaD from other major enterobacterial antiporters. Indeed, Ec-NhaB from E. coli is pHindependent, whereas the activity of Ec-NhaA gradually increases upon pH shift from 7.0 to 8.0, reaching a plateau (9). Curiously, homologous NhaD from Vibrio parahaemolyticus exhibits pH dependence similar to Ec-NhaA rather than Vc-NhaD (31). The Na ϩ /H ϩ antiporters of NhaD type are widely distributed in nature, being found in genomes of pathogenic vibrios, nitrogen-fixing symbionts, magnetotactic cocci and photosynthetic bacteria as well as in higher plants (Fig. 1). In obligate intracellular parasites of Chlamydia genus, NhaD serves as a sole Na ϩ /H ϩ antiporter (32-33). However, very little is known about the molecular mechanisms of cation exchange mediated by these proteins. In the absence of a detailed crystal structure, identification of functionally important residues in antiporters by sitedirected mutagenesis remains one of the most informative approaches. Because Na ϩ /H ϩ antiporters are exchanging cations, negatively charged residues are obvious primary targets for mutagenesis (see for example Refs. 18 and 23). In our previous work we found that mutation of three polar residues, Asp-344, Thr-345 (TMS 3 X and loop IX-X), and Asp-393 (within TMS XI) severely affects the Na ϩ -dependent proton transfer mediated by . In the present study we extended these observations by muta...
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