The role of cysteine residues in the rearrangement of uridine to pseudouridine catalyzed by pseudouridine synthase I.
Escherichia coli tRNA pseudouridine synthase I (PSUI) catalyzes the conversion of uridine residues to pseudouridine in positions 38, 39, and 40 of various tRNA molecules. In previous biochemical studies with this enzyme (Kammen, H. O., Marvel, C. C., Hardy, L., and Penhoet, E. E. (1988) J. Biol. Chem. 263, 2255-2263) it was reported that cysteine residues are important in maintaining the active structure of the enzyme and are possibly involved in the catalytic reaction mechanism via a covalent cysteine intermediate. In order to further investigate the biochemical properties of PSUI, a high level expression and purification system for the enzyme and its corresponding mutants was developed. PSUI has three cysteine residues among 270 amino acids. In the present investigation, each cysteine residue was individually changed to serine and alanine. In addition, a triple mutant was prepared wherein all three cysteine residues were replaced by alanine. Surprisingly, while two of the three cysteine to serine mutants were inactive, all alanine mutants exhibited near wild-type levels of activity, including the triple mutant. These results provide the first direct and unambiguous chemical evidence against a covalent cysteine intermediate in the rearrangement mechanism of uridine to pseudouridine.
Zhao
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Department of Chemistry, Columbia University, New York, New York 10027, USA.
Horne
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D002384:Catalysis; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D019751:Intramolecular Transferases; D007535:Isomerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011560:Pseudouridine / Q000737:chemistry*; D014529:Uridine / Q000737:chemistry*
D011560:Pseudouridine; D007535:Isomerases; D019751:Intramolecular Transferases; C023199:tRNA-pseudouridine synthase I; D003545:Cysteine; D014529:Uridine