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Protein Engineering vol. 16 no. 8 pp. 607-614, 2003
© 2003 Oxford University Press

The structural roles of conserved Pro196, Pro197 and His199 in the mechanism of thymidylate synthase

Dolores Gonzalez-Pacanowska1, Luis M. Ruiz-Perez1, María Angeles Carreras-Gómez1, Maria Paola Costi2, Robert M. Stroud3, Janet Finer-Moore3 and Daniel V. Santi4

Department of Biochemistry and Biophysics and Department of Pharmaceutical Chemistry, S412-B, University of California San Francisco, San Francisco, CA 94143-0448, USA. Present addresses: 1Istituto de Paristologia ‘Lopez Neyra’, Via Ventanilla 14, 18002 Granada, Spain, 2Dipartimento di Scienze Farmaceutiche, Università di Modena e Reggio Emilia, Via Campi 183, 41100 Modena, Italy and 4Kosan Biosciences, 3832 Bay Center Place, Hayward, CA 94545, USA

3 To whom correspondence should be addressed. e-mail: santi{at}socrates.ucsf.edu; finer{at}msg.ucsf.edu

We generated replacement sets for three highly conserved residues, Pro196, Pro197 and His199, that flank the catalytic nucleophile, Cys198. Pro196 and Pro197 have restricted mobility that could be important for the structural transitions known to be essential for activity. To test this hypothesis we obtained and characterized 13 amino acid substitutions for Pro196, 14 for Pro197 and 14 for His199. All of the Pro196 and Pro197 variants, except P197R, and four of the His199 variants complemented TS-deficient Escherichia coli cells, indicating they had at least 1% of wild-type activity. For all His199 mutations, kcat/Km for substrate and cofactor decreased more than 40-fold, suggesting that the conserved hydrogen bond network co-ordinated by His199 is important for catalysis. Pro196 can be substituted with small hydrophilic residues with little loss in kcat, but 15- to 23-fold increases in KmdUMP. Small hydrophobic substitutions for Pro197 were most active, and the most conservative mutant, P197A, had only a 5-fold lower kcat/KmdUMP than wild-type TS. Several Pro196 and Pro197 variants were temperature sensitive. The small effects of Pro196 or Pro197 mutations on enzyme kinetics suggest that the conformational restrictions encoded by the Pro–Pro sequence are largely maintained when either member of the pair is mutated.

Received February 24, 2003; revised June 19, 2003; accepted June 20, 2003.


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