Labeling aromatic residues

More specific evidence came from affinity labeling with molecules which could react with specific amino acid group sat or adjacent to the substrate site. These labels were substrate analogues and competitive inhibitors. Substituted aryl alkyl ketones were used. TV-p-toluene-sulphonyl-L-phenylalanine chloromethyl ketone (TPCK) blocked the activity of chymotrypsin. Subsequent sequence analysis identified histidine 57 as its site of binding (see Hess, 1971, p 213, The Enzymes, 3rd ed.). Trypsin, with its preference for basic rather than aromatic residues adjacent to the peptide bond, was not blocked by TPCK but was susceptible to iV-p-toluenesulphonyl-L-lysine chloromethyl ketone (TLCK) (Keil, ibid, p249). 

The naturally occurring aromatic amino acids phenylalanine, tryptophane and tyrosine (Fig. 1) have been labelled with fluorine-18 through similar electrophilic substitution methods [7]. Aromatic residues contained in peptides have been labelled with CH3C02[ F]F [105,106], an example of direct labelling of macromolecules. However, direct labelling of macromolecules is usually not the method of choice nowadays (see Section 6). 

RNRs catalyze the reduction of ribonucleotides to deoxyribonucleotides, which represents the first committed step in DNA biosynthesis and repair.These enzymes are therefore required for all known life forms. Three classes of RNRs have been identified, all of which turn out to be metalloenzymes. The so-called class I RNRs contain a diiron site (see Cobalt Bn Enzymes Coenzymes and Iron-Sulfur Proteins for the other two types of RNRs). As diagrammed in Figure 5, these enzymes generate first a tyrosyl radical proximal to the diiron site in the protein subunit labeled R2, and then a thiyl radical in an adjacent subunit (Rl) that ultimately abstracts a hydrogen atom from the ribonucleotide substrate. This controlled tyrosine/thiol radical transfer must occur over an estimated distance of 35 A, and a highly choreographed proton-coupled electron transfer (PCET) mechanism across intervening aromatic residues has been proposed. Perhaps, even more remarkably,... 

Figures. NOSEY spectra of aromatic protons of the complex of Y50F with GMP. Interresidue NOEs and the NOEs between the bound GMP and the aromatic residues are numerically labeled 1-7 are the NOEs between a-protons and aromatic protons 8, 10 and 14 are the NOEs between Fb and Fd 9, 11 and 16 are the NOEs between Fb and Yd 12, 13 and 17 are the NOEs between Y77 and W70 15 and 18 are the NOEs between Y78 and F50 19 and 20 are the NOE between H8 of GMP and F50 21 is the NOE between W70 and an amide or aromatic proton 22 and 23 are the NOE between H8 of GMP and Y78.

Figure 8. (A) Overall fold of tan ARO/CYC. (B) Close-up view of binding cavity with a polyketide intermediate docked and hydrophobic aromatic residues labeled. (C) Crystals of ARO/CYC.

In the enzyme acetylcholinesterase, there is evidence that the charged quaternary ammonium group of ACh interacts with an aromatic residue, tryptophan (83). The evidence concerning this interaction is much less certain in the nicotinic receptor, but the putative binding site region contains a number of aromatic amino acids that have been investigated. Several of them are labeled by photoaffinity reagents (84). 

In order to Investigate the aromatic residues In detail It Is necessary to resort to Isotopic substitution methods to provide the required spectral simplification. For this reason we have studied enzymes containing selectively deuterlated aromatic residues and also containing 9f-labelled amino acids. The deuterlatlon approach was pioneered some years ago by Jardetzky (13) and Katz (14) and their colleagues. More recently Sykes et al. (15) and other workers (11, 12, 16, 17, 18) have Incorporated fluorine labelled amino acids Into proteins and examined their NMR spectra. 

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