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Science 7 March 1986:
Vol. 231. no. 4742, pp. 1123 - 1128
DOI: 10.1126/science.3511529
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Articles

Science, Vol 231, Issue 4742, 1123-1128
Copyright © 1986 by American Association for the Advancement of Science

articles

Functional role of aspartic acid-27 in dihydrofolate reductase revealed by mutagenesis

EE Howell, JE Villafranca, MS Warren, SJ Oatley, and J Kraut

The crystal structures and enzymic properties of two mutant dihydrofolate reductases (Escherichia coli) were studied in order to clarify the functional role of an invariant carboxylic acid (aspartic acid at position 27) at the substrate binding site. One mutation, constructed by oligonucleotide-directed mutagenesis, replaces Asp27 with asparagine; the other is a primary-site revertant to Ser27. The only structural perturbations involve two internally bound water molecules. Both mutants have low but readily measurable activity, which increases rapidly with decreasing pH. The mutant enzymes were also characterized with respect to relative folate: dihydrofolate activities and kinetic deuterium isotope effects. It is concluded that Asp27 participates in protonation of the substrate but not in electrostatic stabilization of a positively charged, protonated transition state.


THIS ARTICLE HAS BEEN CITED BY OTHER ARTICLES:
Structural and Kinetic Evidence for an Extended Hydrogen-bonding Network in Catalysis of Methyl Group Transfer: ROLE OF AN ACTIVE SITE ASPARAGINE RESIDUE IN ACTIVATION OF METHYL TRANSFER BY METHYLTRANSFERASES.
T. I. Doukov, H. Hemmi, C. L. Drennan, and S. W. Ragsdale (2007)
J. Biol. Chem. 282, 6609-6618
   Abstract »    Full Text »    PDF »
Identification of Amino Acids Required for the Functional Up-regulation of Human Dihydrofolate Reductase Protein in Response to Antifolate Treatment.
N. Skacel, L. G. Menon, P. J. Mishra, R. Peters, D. Banerjee, J. R. Bertino, and E. E. Abali (2005)
J. Biol. Chem. 280, 22721-22731
   Abstract »    Full Text »    PDF »
Combinatorial exploration of the catalytic site of a drug-resistant dihydrofolate reductase: creating alternative functional configurations.
A. R. Schmitzer, F. Lepine, and J. N. Pelletier (2004)
Protein Eng. Des. Sel. 17, 809-819
   Abstract »    Full Text »    PDF »
Mechanistic Studies of R67 Dihydrofolate Reductase. EFFECTS OF pH AND AN H62C MUTATION.
H. Park, P. Zhuang, R. Nichols, and E. E. Howell (1997)
J. Biol. Chem. 272, 2252-2258
   Abstract »    Full Text »    PDF »
High-resolution epitope mapping of hGH-receptor interactions by alanine-scanning mutagenesis.
B. Cunningham and J. Wells (1989)
Science 244, 1081-1085
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Insights into enzyme function from studies on mutants of dihydrofolate reductase.
S. Benkovic, C. Fierke, and A. Naylor (1988)
Science 239, 1105-1110
   Abstract »    PDF »
Tinkering with enzymes: what are we learning?.
J. Knowles (1987)
Science 236, 1252-1258
   Abstract »    PDF »
Mechanisms of antibody binding to a protein.
E. Getzoff, H. Geysen, S. Rodda, H Alexander, J. Tainer, and R. Lerner (1987)
Science 235, 1191-1196
   Abstract »    PDF »
Implications for Enzymic Catalysis from Free-energy Reaction Coordinate Profiles.
C.A. Fierke, R.D. Kuchta, K.A. Johnson, and S.J. Benkovic (1987)
Cold Spring Harb Symp Quant Biol 52, 631-638
   Abstract »    PDF »
A Vibrational Structure of 7,8-Dihydrobiopterin Bound to Dihydroneopterin Aldolase.
H. Deng, R. Callender, and G. E. Dale (2000)
J. Biol. Chem. 275, 30139-30143
   Abstract »    Full Text »    PDF »
Vibrational Structure of Dihydrofolate Bound to R67 Dihydrofolate Reductase.
H. Deng, R. Callender, and E. Howell (2001)
J. Biol. Chem. 276, 48956-48960
   Abstract »    Full Text »    PDF »


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