The Interaction of Tryptophanyl-tRNA Synthetase with the Triazine Dye Brown MX-5BR

Abstract

Brown MX‐5 BR specifically and irreversibly inactivates tryptophanyl‐tRNA synthetase from Bacillus stearo‐thermophilus at pH 8.5. The enzyme is protected from inactivation by the substrates tryptophan and ATP and to lesser extents by ADP, AMP, the product inorganic pyrophosphate and other nucleotides such as GTP. The Kd of the pure reactive dye for the enzyme uah measured to be 6.7 × 10−5 M. The Km values of the two substrates tryptophan and MgATP were found to be 1 × 10−5 M and 5×10−5 M respectively. The aminated dye is a competitive inhibitor of tryptophanyl‐tRNA synthetase with respect to both tryptophan and MgATP with Ki values of 2×10−4 M against both substrates. The use of this dye as an active‐site‐directed affinity label is discussed.

The isolation of a peptide from the catalytic domain of Bacillus stearothermophilus tryptophyl-tRNA synthetase. The interaction of Brown MX-5BR with tyrosyl-tRNA synthetase

“…80%) of the dye purified by t.l.c. The inactivating ability of pure Brown MX-5BR with respect to WTS has been reported previously (McArdell et al, 1982). Fig.…”

“…3 shows that tyrosine does not protect YTS from inactivation by Brown MX-5BR, whereas ATP confers significant protection to this enzyme. The inhibition constant of aminated Brown MX-5BR with respect to WTS reported previously (McArdell et al, 1982) showed that the dye has a high affinity for the tryptophyladenylate-binding site. The inhibition of YTS by aminated Brown MX-5BR exhibited mixed kinetics and a low affinity for the tyrosine-binding site.…”

The inhibition of glucokinase and glycerokinase from Bacillus stearothermophilus by the triazine dye Procion Blue MX-3G

“…where kobs is the observed rate of enzyme inactivation at a given concentration of the dye, D, k+2 is the maximum rate of inactivation (min-1) and Kd is the apparent dissociation constant, k-,/k+1, of the enzymedye complex (Kitz & Wilson, 1962;Clonis & Lowe, 1980;Witt & Roskoski, 1980;McArdell et al, 1982).…”

“…The accumulated evidence indicates that the polysulphonated aromatic chromophores of the triazine dyes mimic naturally occurring heterocyclic biological molecules such as adenosine phosphates, NAD+, NADP+, acetyl-CoA, folate and flavins (Lowe et al,198 la; Edwards & Woody, 1979;Johnson & Stevenson, 1980;Small et al, 1981;Clonis et al, 1981). The dyes do not bind solely to proteins with a 'dinucleotide fold', as originally suggested (Thompson et al, 1975); for example the elution characteristics of aminoacyl-tRNA synthetases from triazine dye affinity-chromatography columns indicate that some dyes can mimic amino acids and others the tRNA substrates McArdell et al, 1982).…”

Studies on the nature of transition‐metal‐ion‐mediated binding of triazine dyes to enzymes

“…The ability of reactive chlorotriazine dyes to mimic the binding of biological ligands has led to their exploitation as effective irreversible affinity labels, particularly for nucleotidedependent enzymes such as dehydrogenases [lo, 17, 181, aminoacyl-tRNA synthetases [19] and kinases [lo, 15, 201. More recent studies have shown that horse liver alcohol dehydrogenase is irreversibly inactivated by a dichlorotriazinyl analogue of Cibacron blue F3G-A, Procion blue MX-R, and that subsequent isolation of a labelled peptide has shown that the dye is attached to the thiol side chain of Cys-174 in the catalytic domain of the enzyme [IS]. In the present study, the ability of a number of analogues of Procion red H-8BN to inactive carboxypeptidase G-2 in the presence and absence of metal ions was investigated in order to establish a suitable affinity label for this enzyme.…”