THE N-TERMINAL AMINO-ACID-SEQUENCES OF THE FIREFLY LUCIFERASE ARE IMPORTANT FOR THE STABILITY OF THE ENZYME

The structural and catalytic role of the N-terminal amino acid sequenc e of Photinus pyralis luciferase was investigated by site-directed mut agenesis. The firefly luciferase activity of a series of deletion and site-directed mutants in the amino-terminal region was investigated in vitro and in vivo. The mutant luciferases were produced either by in vitro transcription and translation or by expressing the cDNA encoding firefly luciferase in the yeast Saccharomyces cerevisiae as a fusion protein to the galactose DNA binding domain protein. Deleting the N-te rminal amino acid residues from 3 to 10 dramatically reduced the lucif erase activity to less than 1% of the wild-type activity. A marked dec rease in the activity (to <5%) was also observed with the Lys8Glu muta nt. The Gly10Arg and Pro11Ala mutants showed 20-30% activity compared to the wild type. On the other handZ mutant Asn6Lys retained the wild- type activity level. Randomizing 3-11 N-terminal amino acids also show ed a marked decrease in activity (<1%). The mutant luciferases with ex tremely low levels of enzymatic activity were thermally unstable. Thes e mutational and stability data correlate with the crystal structure o f firefly luciferase in which specific amino acids in the N-terminal r egion form hydrogen bonds to the amino acids in the neighboring beta-s tranded sheet. Because the N-terminal region is not part of the active site, the present results suggest that the highly conserved N-termina l amino acid sequences of the firefly luciferase are important for sta bilizing the protein in its proper conformation for optimal enzyme act ivity.