PHYSICOCHEMICAL PROPERTIES OF RECOMBINANT LUCIOLA-MINGRELICA-LUCIFERASE AND ITS MUTANT FORMS

Physicochemical properties of recombinant L. mingrelica luciferase syn thesized by E. coli cells were studied. The catalytic and spectral pro perties of the recombinant luciferase were similar to those of the nat ive one, but the former was less stable due to the presence of an addi tional Cys residue. Mutant forms of L. mingrelica firefly luciferase w ith point mutations Cys-82-->Ala, Cys-260-->Ala, Cys-393-->Ala, and Th r-204-->Asp were constructed using the method of site-specific mutagen esis. Cys-82,260,393-->Ala mutations changed slightly the K-m for ATP and luciferin but did not influence k(cat). The Cys-393-->Ala mutant a ppeared to be more stable than the native luciferase. Mutation Thr-204 -->Asp resulted in a 8-fold increase in the ATP binding constant and a 2-fold increase in k(cat), indicating that Thr-204 may be located in the ATP-binding region of the luciferase. Dithiothreitol, ethylene gly col, bovine serum albumin, and trehalose had a stabilizing effect on t he native, recombinant, and mutant luciferases.