The active-site cysteines of firefly luciferase: Chemical modification and mutagenesis studies.

摘要

Firefly luciferase catalyses the emission of light from substrates ATP and D-luciferin by a two step process requiring molecular oxygen. The emission spectrum is influenced by pH. Normal yellow-green emission ({dollar}lambda{dollar}max = 562 nm) is observed at alkaline pH, with a quantum yield near unity, while acidic conditions result in red emission ({dollar}lambda{dollar}max = 615 nm), quantum yield 0.33. Previous results have suggested that a cysteine residue in the active-site, acting as a general based, may be responsible for this pH dependent effect on the emission.; The primary structure of firefly luciferase contains four cysteine residues, two of which are protected from reaction with sulfhydryl modifying reagents such as DTNB and NEM in the presence of Mg{dollar}sp*{dollar}ATP and dehydroluciferin. Using ({dollar}sp{lcub}14{rcub}{dollar}C) -NEM, and taking advantage of this differential modification in the presence and absence of substrates, active-center cystiene containing tryptic peptides were isolated by reverse phase HPLC and subjected to automated gas phase sequencing. These residues were identified as Cys216 and Cys391 by comparison to the predicted primary sequence deduced from the nucleotide sequence of the luc cDNA.; The purification of luciferase from an overexpressing E. coli host was undertaken to facilitate comparison to the native enzyme isolated from firefly lanterns. After extraction by French Press and ammonium sulfate fractionation, the enzyme was further purified by FPLC hydrophobic interaction and gel filtration chromatography. This provided a sample of sufficient specific activity to allow crystallization. This resulted in an enzyme preparation with specific activity identical to that obtainable by purification from native sources. The overall yield of this preparation was 20%.; In order to assess the role of the active-center cysteines in the pH dependent control of the emission spectrum, oligonucleotide directed mutagenesis of the luc cDNA was employed to individually replace active-center cystienes with alanine. The two mutant luciferases, C216A and C391A were purified using the procedure described above and characterized according to the kinetics and spectrum of light emission. C391A displayed a two fold increase in Km for luciferin and both mutants displayed a two fold increase in maximum intensity measured under steady state conditions. No significant difference in emission spectrum at pH 8 was detected for either mutant indicating that neither of the active-center cysteine residues is acting as the general base postulated to be responsible for control of the emission spectrum in firefly luciferase.