The roles of the essential Asp-48 and highly conserved His-43 elucidated by the pH dependence of the pseudouridine synthase TruB.
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.
Hamilton
Christopher S
CS
Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716, USA.
Spedaliere
Christopher J
CJ
Ginter
Joy M
JM
Johnston
Murray V
MV
Mueller
Eugene G
EG
D001224:Aspartic Acid / Q000737:chemistry / Q000235:genetics*; D020029:Base Pairing; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D014644:Genetic Variation; D006639:Histidine / Q000737:chemistry / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D019753:Intramolecular Lyases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D019751:Intramolecular Transferases; D007700:Kinetics; D008958:Models, Molecular; D008961:Models, Structural; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D013379:Substrate Specificity; D014443:Tyrosine / Q000737:chemistry; D014529:Uridine / Q000737:chemistry
D029968:Escherichia coli Proteins; D001224:Aspartic Acid; D014443:Tyrosine; D006639:Histidine; D019751:Intramolecular Transferases; C096995:pseudouridine synthases; D019753:Intramolecular Lyases; D014529:Uridine