News and Publications

Nuclear Magnetic Resonance Studies of the Structure and Mechanism of Enzymes

H.J. Dyson, P.E. Wright, J. Chung, G. Kroon, M. Martinez-Yamout, M.J. Osborne, U. Rudsander, S. Scrofani, B. Xia, E. Zaborowski, L.L. Tennant, S.J. Benkovic,* A. Holmgren**

* Pennsylvania State University, University Park, PA
** Karolinska Institute, Stockholm, Sweden

We use site-specific information from nuclear magnetic resonance (NMR) studies to further the understanding of enzyme function through study of structure and dynamics. Studies focus on the mechanism of enzymes and catalytic antibodies and the relationship between dynamics and function.

DYNAMICS IN ENZYME ACTION

We are exploring the relationship between dynamics of the polypeptide chain and enzyme catalysis. We hypothesize that efficient enzyme catalysis requires flexibility of the active site and that enzymes have therefore evolved to incorporate this flexibility. To test these hypotheses, we are using mutagenesis and detailed characterization of changes in enzymatic function, structure, and dynamics in 2 extremely different enzyme systems: dihydrofolate reductase, for which we already have clear evidence that dynamics play a role in catalysis, and a metallo-ß-lactamase, for which the crystal structure suggests flexibility in the active-site region (Fig. 1).

NMR relaxation measurements on substrate and cofactor complexes of dihydrofolate reductase, in collaboration with S. Benkovic, Pennsylvania State University, are providing novel insights into the relationship between dynamics and enzyme activity. Motions on a wide range of timescales (picoseconds to milliseconds) have been detected and can be correlated with enzyme function. Both enzymes are clinically important drug targets; dihydrofolate reductase is an anticancer drug, and the lactamase is used to combat antibiotic resistance.

DESIGN AND SYNTHESIS OF PROTEINS

We have developed an efficient method of overproducing proteins for NMR that involves designing and synthesizing genes specifically for expression in Escherichia coli. This method is exceptionally useful in the production of several proteins, including Thiobacillus ferrooxidans rusticyanin and soybean leghemoglobin. It is also easily adaptable for the production of site-specific mutants, an important aspect of the investigation of the relationship between structure and function.

DESIGN OF A CATALYTIC ANTIBODY

One aspect of our work on the design of catalysts is a long-standing project on the characterization and redesign of the Fv fragment of a catalytic antibody. This work is extremely promising for understanding the influence of structure on function for enzymes. Because they have evolved over millions of years, most enzymes are exquisitely tuned to the reactions they catalyze and may also be tolerant of mutations. In contrast, catalytic antibodies have much lower efficiency and specificity. Knowledge of the local structure and dynamics of the catalytic site will allow novel insights into the mechanisms of antibody catalysis and will guide future work aimed at enhancing the catalytic efficiency. These studies will also provide valuable insights into the structural and functional evolution of enzymes. The Fv fragment of the catalytic antibody 43C9 has been expressed and labeled uniformly with ²H, ¹³C, and ¹⁵N, and backbone resonance assignments are nearing completion.

THIOREDOXIN

Thioredoxin, a small,108-residue thiol-disulfide oxidoreductase, has many functions in the cell, including reduction of ribonucleotides to form deoxyribonucleotides for DNA synthesis. Thioredoxins occur in all living organisms and in viruses. Mammalian thioredoxins have a role in cellular control mechanisms and have been implicated in human diseases; serum levels of thioredoxin are elevated in AIDS patients. One of the primary functions of thioredoxin in the cell is as a protein disulfide reductase, a function vital for the prevention of misfolded proteins in vivo.

The E coli thioredoxin system has been fully characterized by NMR, including the calculation of high-resolution structures for both the oxidized (disulfide) and the reduced (dithiol) forms of the protein. Using backbone dynamics and amide proton hydrogen exchange, we found that functional differences in phage systems between oxidized and reduced thioredoxin were due to differences in the flexibility of the molecules, rather than to structural differences. We also discovered that the reduction reaction of thioredoxin depends on the movement of protons during the 2-electron--2-proton transfer reaction as a substrate disulfide is reduced. An extensive series of experiments on the pH-dependence of the NMR spectrum provided important insights into the complex relationship between the mechanism of action of this enzyme and local structure at the active site, including the presence of conserved, buried charged residues. This project has been extended to include a structural NMR study of a related protein, glutaredoxin 2, which shows only limited sequence similarity to other thioredoxins and glutaredoxins and has a higher molecular weight.

PUBLICATIONS

Bender, C.J., Casimiro, D., Dyson, H.J. Electron spin envelope modulation spectra of Thiobacillus ferrooxidans rusticyanin and a mutant lacking one of the copper ligands. J. Chem. Soc. (Faraday) 93:3967, 1997.

Casimiro, D., Wright, P.E., Dyson, H.J. PCR-based gene synthesis for protein overproduction. Structure 5:1407, 1997.

Dillet, V., Dyson, H.J., Bashford, D. Calculations of electrostatic interactions and pK_as in the active site of Escherichia coli thioredoxin. Biochemistry, in press.

Jeng, M.-F., Reymond, M.T., Tennant, L.L., Holmgren, A., Dyson, H.J. NMR characterization of a single-cysteine mutant of Escherichia coli thioredoxin and a covalent thioredoxin-peptide complex. Eur. J. Biochem., in press.

Scrofani, S.D.B., Wright, P.E., Dyson, H.J. ¹H, ¹³C, and ¹⁵N NMR backbone assignments of the 25.5 kDa metallo-ß-lactamase from Bacteroides fragilis. J. Biomol. NMR, in press.

Scrofani, S.D.B., Wright, P.E., Dyson, H.J. The identification of metal-binding ligand residues in metalloproteins using nuclear magnetic resonance spectroscopy. Protein Sci., in press.

Zhu, L., Dyson, H.J., Wright, P.E. A NOESY-HSQC simulation program SPIRIT. J. Biomol. NMR 11:17, 1997.