Mutation of a protease-sensitive region in firefly luciferase alters light emission properties.
Luciferase (EC 1.13.12.7) from the North American firefly, Photinus pyralis, is widely used as a reporter enzyme in cell biology. One of its distinctive properties is a pronounced susceptibility to proteolytic degradation that causes luciferase to have a very short intracellular half-life. To define the structural basis for this behavior and possibly facilitate the design of more stable forms of luciferase, limited proteolysis studies were undertaken using trypsin and chymotrypsin to identify regions of the protein whose accessible and flexible character rendered them especially sensitive to cleavage. Regions of amino acid sequence 206-220 and 329-341 were found to be sensitive, and because the region around 206-220 had high homology with other luciferases, CoA ligases, and peptidyl synthetases, this region was selected for mutagenesis experiments intended to determine which of its amino acids were essential for activity. Surprisingly, many highly conserved residues including Ser198, Ser201, Thr202, and Gly203 could be mutated with little effect on the luminescent activity of P. pyralis luciferase. One mutation, however, S198T, caused several alterations in enzymatic properties including shifting the pH optimum from 8.1 to 8.7, lowering the Km for Mg-ATP by a factor of 4 and increasing the half-time for light emission decay by a factor of up to 150. While the S198T luciferase was less active than wild type, activity could be restored by the introduction of the additional L194F and N197Y mutations. In addition to indicating the involvement of this region in ATP binding, these results provide a new form of the enzyme that affords a more versatile reporter system.
Thompson
J F
JF
Molecular Sciences Department, Central Research Division, Pfizer Inc., Groton, Connecticut 06340, USA. thompsonf@pfizer.com
Geoghegan
K F
KF
Lloyd
D B
DB
Lanzetti
A J
AJ
Magyar
R A
RA
Anderson
S M
SM
Branchini
B R
BR
D000595:Amino Acid Sequence; D000818:Animals; D001517:Coleoptera; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008156:Luciferases / Q000737:chemistry / Q000235:genetics*; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D009154:Mutation; D017386:Sequence Homology, Amino Acid
D008156:Luciferases