RESUMO
All known pseudouridine synthases have a conserved aspartic acid residue that is essential for catalysis, Asp-48 in Escherichia coli TruB. To probe the role of this residue, inactive D48C TruB was oxidized to generate the sulfinic acid cognate of aspartic acid. The oxidation restored significant but reduced catalytic activity, consistent with the proposed roles of Asp-48 as a nucleophile and general base. The family of pseudouridine synthases including TruB also has a nearly invariant histidine residue, His-43 in the E. coli enzyme. To examine the role of this conserved residue, site-directed mutagenesis was used to generate H43Q, H43N, H43A, H43G, and H43F TruB. Except for phenylalanine, the substitutions seriously impaired the enzyme, but all of the altered TruB retained significant activity. To examine the roles of Asp-48 and His-43 more fully, the pH dependences of wild-type, oxidized D48C, and H43A TruB were determined. The wild-type enzyme displays a typical bell-shaped profile. With oxidized D48C TruB, logk(cat) varies linearly with pH, suggesting the participation of specific rather than general base catalysis. Substitution of His-43 perturbs the pH profile, but it remains bell-shaped. The ascending limb of the pH profile is assigned to Asp-48, and the descending limb is tentatively ascribed to an active site tyrosine residue, the bound substrate uridine, or the bound product pseudouridine.
Assuntos
Ácido Aspártico/genética , Proteínas de Escherichia coli/química , Histidina/genética , Liases Intramoleculares/química , Liases Intramoleculares/genética , Ácido Aspártico/química , Pareamento de Bases , Sítios de Ligação/genética , Catálise , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Variação Genética , Histidina/química , Concentração de Íons de Hidrogênio , Liases Intramoleculares/isolamento & purificação , Liases Intramoleculares/metabolismo , Transferases Intramoleculares , Cinética , Modelos Moleculares , Modelos Estruturais , Mutagênese Sítio-Dirigida , Oxirredução , Conformação Proteica , Estrutura Secundária de Proteína , Especificidade por Substrato , Tirosina/química , Uridina/químicaRESUMO
RNA containing 5-fluorouridine, [f 5U]RNA, has been used as a mechanistic probe for the pseudouridine synthases, which convert uridine in RNA to its C-glycoside isomer, pseudouridine. Hydrated products of f 5U were attributed to ester hydrolysis of a covalent complex between an essential aspartic acid residue and f 5U, and the results were construed as strong support for a mechanism involving Michael addition by the aspartic acid residue. Labeling studies with [18O]water are now reported that rule out such ester hydrolysis in one pseudouridine synthase, TruB. The aspartic acid residue does not become labeled, and the hydroxyl group in the hydrated product of f 5U derives directly from solvent. The hydrated product, therefore, cannot be construed to support Michael addition during the conversion of uridine to pseudouridine, but the results do not rule out such a mechanism. A hypothesis is offered for the seemingly disparate behavior of different pseudouridine synthases toward [f 5U]RNA.
Assuntos
Hidroliases/metabolismo , Pseudouridina/biossíntese , Uridina/análogos & derivados , Escherichia coli/enzimologia , Escherichia coli/genética , Hidroliases/genética , Hidrólise , RNA/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Uridina/metabolismoRESUMO
RNA containing 5-fluorouridine has been assumed to inhibit strongly or irreversibly the pseudouridine synthases that act on the RNA. RNA transcripts containing 5-fluorouridine in place of uridine have, therefore, been added to reconstituted systems in order to investigate the importance of particular pseudouridine residues in a given RNA by inactivating the pseudouridine synthase responsible for their generation. In sharp contradiction to the assumption of universal inhibition of pseudouridine synthases by RNA containing 5-fluorouridine, the Escherichia coli pseudouridine synthase TruB, which has physiologically critical eukaryotic homologs, is not inhibited by such RNA. Instead, the RNA containing 5-fluorouridine was handled as a substrate by TruB. The E. coli pseudouridine synthase RluA, on the other hand, forms a covalent complex and is inhibited stoichiometrically by RNA containing 5-fluorouridine. We offer a hypothesis for this disparate behavior and urge caution in interpreting results from reconstitution experiments in which RNA containing 5-fluorouridine is assumed to inhibit a pseudouridine synthase, as normal function may result from a failure to inactivate the targeted enzyme rather than from the absence of nonessential pseudouridine residues.
