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. 1996 Aug;5(8):1523–1530. doi: 10.1002/pro.5560050808

A catalytic function for the structurally conserved residue Phe 100 of ribonuclease T1.

J Doumen 1, M Gonciarz 1, I Zegers 1, R Loris 1, L Wyns 1, J Steyaert 1
PMCID: PMC2143497  PMID: 8844843

Abstract

The function of the conserved Phe 100 residue of RNase T1 (EC 3.1.27.3) has been investigated by site-directed mutagenesis and X-ray crystallography. Replacement of Phe 100 by alanine results in a mutant enzyme with kcat reduced 75-fold and a small increase in Km for the dinucleoside phosphate substrate GpC. The Phe 100 Ala substitution has similar effects on the turnover rates of GpC and its minimal analogue GpOMe, in which the leaving cytidine is replaced by methanol. The contribution to catalysis is independent of the nature of the leaving group, indicating that Phe 100 belongs to the primary site. The contribution of Phe 100 to catalysis may result from a direct van der Waals contact between its aromatic ring and the phosphate moiety of the substrate. Phe 100 may also contribute to the positioning of the pentacovalent phosphorus of the transition state, relative to other catalytic residues. If compared to the corresponding wild-type data, the structural implications of the mutation in the present crystal structure of Phe 100 Ala RNase T1 complexed with the specific inhibitor 2'-GMP are restricted to the active site. Repositioning of 2'-GMP, caused by the Phe 100 Ala mutation, generates new or improved contacts of the phosphate moiety with Arg 77 and His 92. In contrast, interactions with the Glu 58 carboxylate appear to be weakened. The effects of the His 92 Gln and Phe 100 Ala mutations on GpC turnover are additive in the corresponding double mutant, indicating that the contribution of Phe 100 to catalysis is independent of the catalytic acid His 92. The present results lead to the conclusion that apolar residues may contribute considerably to catalyze conversions of charged molecules to charged products, involving even more polar transition states.

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Selected References

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  1. Ackers G. K., Smith F. R. Effects of site-specific amino acid modification on protein interactions and biological function. Annu Rev Biochem. 1985;54:597–629. doi: 10.1146/annurev.bi.54.070185.003121. [DOI] [PubMed] [Google Scholar]
  2. Arni R., Heinemann U., Tokuoka R., Saenger W. Three-dimensional structure of the ribonuclease T1 2'-GMP complex at 1.9-A resolution. J Biol Chem. 1988 Oct 25;263(30):15358–15368. [PubMed] [Google Scholar]
  3. Carter P. J., Winter G., Wilkinson A. J., Fersht A. R. The use of double mutants to detect structural changes in the active site of the tyrosyl-tRNA synthetase (Bacillus stearothermophilus). Cell. 1984 Oct;38(3):835–840. doi: 10.1016/0092-8674(84)90278-2. [DOI] [PubMed] [Google Scholar]
  4. Eckstein F., Schulz H. H., Rüterjans H., Haar W., Maurer W. Stereochemistry of the transesterification step of ribonuclease T 1 . Biochemistry. 1972 Sep 12;11(19):3507–3512. doi: 10.1021/bi00769a002. [DOI] [PubMed] [Google Scholar]
  5. Fersht A. R. Relationships between apparent binding energies measured in site-directed mutagenesis experiments and energetics of binding and catalysis. Biochemistry. 1988 Mar 8;27(5):1577–1580. doi: 10.1021/bi00405a027. [DOI] [PubMed] [Google Scholar]
  6. Grunert H. P., Zouni A., Beineke M., Quaas R., Georgalis Y., Saenger W., Hahn U. Studies on RNase T1 mutants affecting enzyme catalysis. Eur J Biochem. 1991 Apr 10;197(1):203–207. doi: 10.1111/j.1432-1033.1991.tb15900.x. [DOI] [PubMed] [Google Scholar]
  7. Heinemann U., Saenger W. Specific protein-nucleic acid recognition in ribonuclease T1-2'-guanylic acid complex: an X-ray study. Nature. 1982 Sep 2;299(5878):27–31. doi: 10.1038/299027a0. [DOI] [PubMed] [Google Scholar]
  8. Jones T. A., Zou J. Y., Cowan S. W., Kjeldgaard M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr A. 1991 Mar 1;47(Pt 2):110–119. doi: 10.1107/s0108767390010224. [DOI] [PubMed] [Google Scholar]
  9. Knowles J. R. Enzyme catalysis: not different, just better. Nature. 1991 Mar 14;350(6314):121–124. doi: 10.1038/350121a0. [DOI] [PubMed] [Google Scholar]
  10. Koepke J., Maslowska M., Heinemann U., Saenger W. Three-dimensional structure of ribonuclease T1 complexed with guanylyl-2',5'-guanosine at 1.8 A resolution. J Mol Biol. 1989 Apr 5;206(3):475–488. doi: 10.1016/0022-2836(89)90495-6. [DOI] [PubMed] [Google Scholar]
  11. Meiering E. M., Serrano L., Fersht A. R. Effect of active site residues in barnase on activity and stability. J Mol Biol. 1992 Jun 5;225(3):585–589. doi: 10.1016/0022-2836(92)90387-y. [DOI] [PubMed] [Google Scholar]
  12. Merritt E. A., Murphy M. E. Raster3D Version 2.0. A program for photorealistic molecular graphics. Acta Crystallogr D Biol Crystallogr. 1994 Nov 1;50(Pt 6):869–873. doi: 10.1107/S0907444994006396. [DOI] [PubMed] [Google Scholar]
  13. Nicholls A., Sharp K. A., Honig B. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins. 1991;11(4):281–296. doi: 10.1002/prot.340110407. [DOI] [PubMed] [Google Scholar]
  14. Nishikawa S., Morioka H., Kim H. J., Fuchimura K., Tanaka T., Uesugi S., Hakoshima T., Tomita K., Ohtsuka E., Ikehara M. Two histidine residues are essential for ribonuclease T1 activity as is the case for ribonuclease A. Biochemistry. 1987 Dec 29;26(26):8620–8624. doi: 10.1021/bi00400a019. [DOI] [PubMed] [Google Scholar]
  15. Stanssens P., Opsomer C., McKeown Y. M., Kramer W., Zabeau M., Fritz H. J. Efficient oligonucleotide-directed construction of mutations in expression vectors by the gapped duplex DNA method using alternating selectable markers. Nucleic Acids Res. 1989 Jun 26;17(12):4441–4454. doi: 10.1093/nar/17.12.4441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Steyaert J., Hallenga K., Wyns L., Stanssens P. Histidine-40 of ribonuclease T1 acts as base catalyst when the true catalytic base, glutamic acid-58, is replaced by alanine. Biochemistry. 1990 Sep 25;29(38):9064–9072. doi: 10.1021/bi00490a025. [DOI] [PubMed] [Google Scholar]
  17. Steyaert J., Wyns L. Functional interactions among the His40, Glu58 and His92 catalysts of ribonuclease T1 as studied by double and triple mutants. J Mol Biol. 1993 Feb 5;229(3):770–781. doi: 10.1006/jmbi.1993.1078. [DOI] [PubMed] [Google Scholar]
  18. Steyaert J., Wyns L., Stanssens P. Subsite interactions of ribonuclease T1: viscosity effects indicate that the rate-limiting step of GpN transesterification depends on the nature of N. Biochemistry. 1991 Sep 3;30(35):8661–8665. doi: 10.1021/bi00099a024. [DOI] [PubMed] [Google Scholar]
  19. Wolfenden R., Radzicka A. On the probability of finding a water molecule in a nonpolar cavity. Science. 1994 Aug 12;265(5174):936–937. doi: 10.1126/science.8052849. [DOI] [PubMed] [Google Scholar]
  20. Zegers I., Maes D., Dao-Thi M. H., Poortmans F., Palmer R., Wyns L. The structures of RNase A complexed with 3'-CMP and d(CpA): active site conformation and conserved water molecules. Protein Sci. 1994 Dec;3(12):2322–2339. doi: 10.1002/pro.5560031217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Zegers I., Verhelst P., Choe H. W., Steyaert J., Heinemann U., Saenger W., Wyns L. Role of histidine-40 in ribonuclease T1 catalysis: three-dimensionalstructures of the partially active His40Lys mutant. Biochemistry. 1992 Nov 24;31(46):11317–11325. doi: 10.1021/bi00161a009. [DOI] [PubMed] [Google Scholar]

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