International
Tables for
Crystallography
Volume F
Crystallography of biological macromolecules
Edited by M. G. Rossmann and E. Arnold

International Tables for Crystallography (2006). Vol. F, ch. 12.1, p. 250   | 1 | 2 |

Section 12.1.4. Amino acids as ligands

D. Carvin,a S. A. Islam,b M. J. E. Sternbergb and T. L. Blundellc*

aBiomolecular Modelling Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Field, London WC2A 3PX, England,bInstitute of Cancer Research, 44 Lincoln's Inn Fields, London WC2A 3PX, England, and cDepartment of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge, CB2 1GA, England
Correspondence e-mail:  tom@cryst.bioc.cam.ac.uk

12.1.4. Amino acids as ligands

| top | pdf |

The reactivity of the heavy-atom reagent will also depend on the state of the amino-acid residues in the protein.

The thiolate anion of cysteine, a potent nucleophile, reacts almost irreversibly with mercuric complexes or organomercurials. It also acts as a fast-entering attacking group in SN2 ligand substitution reactions with other class B metals (e.g. Ag, Ir, Rh, Pt, Pd, Au), forming stable complexes. Below pH 6, the thiolate anion becomes protonated. As covalent reactions are less sensitive to hydrogen-ion concentration than ligand substitution reactions, cysteines still bind rapidly with mercurials, but there is negligible reaction with other class B metals (Petsko et al., 1978[link]).

Cystines are very weakly reactive in ligand substitution reactions. However, [\hbox{PtCl}_{4}^{2-}] binds to disulfides in some proteins with displacement of a chloride ion (Lipscomb et al., 1970[link]; Sigler et al., 1968[link]). Mercurials rarely insert spontaneously into disulfide linkages. However, substitution of mercury can be achieved either by the prior application of a reducing agent such as dithiothreitol (Ely et al., 1973[link]; Sperling et al., 1969[link]), or by direct application of reducing mercurous ions (Sperling & Steinberg, 1974[link]).

The non-ionizable thioether group of methionine is unreactive towards mercurials, but the lone pair of electrons on sulfur allows nucleophilic SN2 ligand substitution. Methionine will displace Cl, I, Br and NO2 ligands from platinum complexes to form a stable bond. The reaction of methionine with platinum compounds is not pH sensitive within the normal range. The residue may become unreactive through oxidation, first to the sulfoxide and then to the sulfone; only the sulfoxide can be reduced readily by thiols or other reducing agents.

Below pH 6, histidine exists mainly as an imidazolium cation. Although this is not reactive as a nucleophile, it can interact electrostatically with anionic complexes. At pH 7 and above, the unprotonated imidazole is a good nucleophile, being able to displace Cl, Br, I and NO2 ligands from platinum, silver, mercury and gold complexes. Electrophilic substitution of iodine in the imidazole ring is feasible, but the conditions are severe and it has not proved very useful in preparing derivatives.

At pH < 8.5, the [epsilon]-amino group of lysine is protonated, allowing it to form weak electrostatic interactions with anionic heavy-atom complexes, but not to participate in SN2 substitution reactions. Above pH 9, the free amino group can displace Cl but not Br, I or NO2 ligands from platinum and gold complexes. The pKa of the guanidinium group of arginine is very high (> 12 in proteins), so it interacts electrostatically as a cation with heavy-atom anionic complexes.

The indole ring of tryptophan is relatively inert to electrophilic substitution by iodine, but the ring nitrogen can be mercurated (Tsernoglou & Petsko, 1976[link]). The reaction is not pH dependent, but there should be no competing nucleophiles in the mother liquor. Tryptophan does not usually participate as a ligand in substitution of heavy-atom complexes.

The phenolate oxygen anion of tyrosine is a good nucleophile and has the potential to bind a substantial number of heavy-atom complexes via SN2 ligand substitution reactions. However, it has a very high pKa value of 10.5. Below pH 10, the protonated oxygen predominates, making electrophilic aromatic substitution by iodine the principal reaction.

Aspartic and glutamic acids have side-chain pKa values in the range 3 to 4. At low pH, they will be protonated and unreactive. Above pH 5, the side chains will be anionic, making them good ligands for class A cations such as uranyl and rare earths. Glutamine and asparagine take part in metal coordination but rarely bind strongly enough to metal ligands on their own.

Hydroxyl groups of serines and threonines are fully protonated at normal pH values and are consequently not reactive nucleophiles. Abnormally reactive serines, usually at the active site as in serine proteases and β-lactamases, can react with heavy-atom reagents to give useful derivatives.

References

Ely, K. R., Girling, R. L., Schiffer, M., Cunningham, D. E. & Edmundson, A. B. (1973). Preparation and properties of crystals of a Bence–Jones dimer with mercury inserted into the interchain disulphide bond. Biochemistry, 12, 4233–4237.Google Scholar
Lipscomb, W. N., Reeke, G. N., Hartsuck, J. A., Quiocho, F. A. & Bethge, P. H. (1970). The structure of carboxypeptidase a. VIII. Atomic interpretation at 0.2 nm resolution, a new study of the complex of glycyl-L-tyrosine with CPA, and mechanistic deductions. Philos. Trans. R. Soc. London Ser. B, 257, 177–214.Google Scholar
Petsko, G. A., Phillips, D. C., Williams, R. J. P. & Wilson, I. A. (1978). On the protein crystal chemistry of chloroplatinite ions: general principles and interactions with triose phosphate isomerase. J. Mol. Biol. 120, 345–359.Google Scholar
Sigler, P. B., Blow, D. M., Matthews, B. W. & Henderson, R. (1968). Structure of crystalline alpha-chymotrypsin II. A preliminary report including a hypothesis for the activation mechanism. J. Mol. Biol. 35, 143–164.Google Scholar
Sperling, R., Burstein, Y. & Steinberg, I. Z. (1969). Selective reduction and mercuration of cysteine IV–V in bovine pancreatic ribonuclease. Biochemistry, 8, 3810–3820.Google Scholar
Sperling, R. & Steinberg, I. Z. (1974). Simultaneous reduction and mercuration of disulphide bond A6–A11 of insulin by monovalent mercury. Biochemistry, 13, 2007–2013.Google Scholar
Tsernoglou, D. & Petsko, G.-A. (1976). The crystal structure of a post-synaptic neurotoxin from sea snake at 2.2 Å resolution. FEBS Lett. 68, 1–4.Google Scholar








































to end of page
to top of page