Site-directed mutagenesis of bacterial luciferase: analysis of the 'essential' thiol.
It has been appreciated for many years that the luciferase from the luminous marine bacterium Vibrio harveyi has a highly reactive cysteinyl residue which is protected from alkylation by binding of flavin. Alkylation of the reactive thiol, which resides in a hydrophobic pocket, leads to inactivation of the enzyme. To determine conclusively whether the reactive thiol is required for the catalytic mechanism, we have constructed a mutant by oligonucleotide directed site-specific mutagenesis in which the reactive cysteinyl residue, which resides at position 106 of the alpha subunit, has been replaced with a seryl residue. The resulting alpha 106Ser luciferase retains full activity in the bioluminescence reaction, although the mutant enzyme has a ca 100-fold increase in the FMNH2 dissociation constant. The alpha 106Ser luciferase is still inactivated by N-ethylmaleimide, albeit at about 1/10 the rate of the wild-type (alpha 106Cys) enzyme, demonstrating the existence of a second, less reactive, cysteinyl residue that was obscured in the wild-type enzyme by the highly reactive cysteinyl residue at position alpha 106. An alpha 106Ala variant luciferase was also active, but the alpha 106Val mutant enzyme was about 50-fold less active than the wild type. All three variants (Ser, Ala and Val) appeared to have somewhat reduced affinities for the aldehyde substrate, the valine mutant being the most affected. It is interesting to note that the alpha 106 mutant luciferases are much less subject to aldehyde substrate inhibition than is the wild-type V. harveyi luciferase, suggesting that the molecular mechanism of aldehyde substrate inhibition involves the Cys at alpha 106.
Baldwin
T O
TO
Department of Biochemistry and Biophysics, Texas A&M University, College Station.
Chen
L H
LH
Chlumsky
L J
LJ
Devine
J H
JH
Ziegler
M M
MM
D000447:Aldehydes / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D003545:Cysteine; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005486:Flavin Mononucleotide / Q000378:metabolism; D008156:Luciferases / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009154:Mutation; D014733:Vibrio / Q000201:enzymology* / Q000235:genetics
D000447:Aldehydes; D005486:Flavin Mononucleotide; D008156:Luciferases; D003545:Cysteine