Sequential chemical modification of a histidyl and a cysteinyl residue in bacterial luciferase

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Abstract
Sequential modification of 2 amino acid residues (a histidyl and a cysteinyl residue), both essential for the enzymatic function of bacterial luciferase from Beneckea harveyi, was conducted to determine if the inactivation arising from the chemical modification of either of these residues is due to a conformational change. The modification of the histidyl or cysteinyl residue did not affect the reactivity of the remaining essential residue, suggesting that chemical modification had not caused a change in conformation. Since substrates [FMNH or aldehyde] protect luciferase against inactivation due to modification of either of these residues, it was possible to determine if the initial modification of the histidyl or cysteinyl residue prevented substrate binding by conducting the modification of the remaining residue (i.e., the cysteinyl or histidyl residue, respectively) in the presence of substrates. After modification of the histidyl residue, substrates no longer protected the cysteinyl residue against modification, but after modification of the cysteinyl residue, substrates still protected the histidyl residue against modification. Apparently, the histidyl residue and not the cysteinyl residue of luciferase is essential for the binding of substrates in the bacterial bioluminescent reaction.