The modification with tetranitromethane of an essential tyrosine in the active site of pig fumarase.
Modification of pig heart fumarase (L-malate hydro-lyase, EC 4.2.1.2) with tetranitromethane results in loss of enzymatic activity. The inactivation is slowed down in the presence of substrates, indicating that the modification reaction takes place at the level of the substrate binding sites. From these inactivation kinetics, a value Kd = 78 microM is calculated for the mixture of substrates (L-malate + fumarate). This is in fairly good agreement with the Michaelis constant Km = 31 microM. Spectrophotometric data indicate that modification of one tyrosine residue per fumarase subunit is responsible for the inactivation; one or more additional residues, which do not participate in the binding sites, are modified at much lower rates. Amino acid analyses confirm the presence of nitrotyrosine and exclude the possibility of tetranitromethane-mediated polymerization side-reactions. It is concluded from the pH-dependence of the nitration reaction that the inactivation of fumarase is not caused by cysteine modification. Additional studies of nitration of melittin, a tryptophan-containing model peptide, are described. From the absorption spectra of modified melittin, in comparison with the spectra of nitrofumarase, it is concluded that the tryptophan residues of the latter enzyme remain intact during the reaction with tetranitromethane. Finally, evidence is given for an independent action of the four fumarase subunits, i.e., inactivation of one subunit does not influence the catalysis by the other three subunits. Moreover, it is shown that only fumarase tetramers with all four subunits nitrated are unable to bind to a Sepharose-pyromellitic acid affinity column.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D005649:Fumarate Hydratase / Q000378:metabolism*; D007700:Kinetics; D008697:Methane / Q000031:analogs & derivatives*; D009206:Myocardium / Q000201:enzymology*; D013552:Swine; D013774:Tetranitromethane / Q000494:pharmacology*; D014443:Tyrosine
D000596:Amino Acids; D014443:Tyrosine; D005649:Fumarate Hydratase; D013774:Tetranitromethane; D008697:Methane