Proteolytic inactivation of luciferases from three species of luminous marine bacteria, Beneckea harveyi, Photobacterium fischeri, and Photobacterium phosphoreum: evidence of a conserved structural feature.

Abstract

Upon limited proteolysis of luciferases from the luminous marine bacteria P. fischeri, P. phosphoreum and B. harveyi, the rate of loss of luciferase activity is the same as the rate of loss of the heavier subunit of all 3 enzymes. It thus appears that the larger subunit of the luciferase from P. phosphoreum should be designated .alpha. based on its apparent homology with the .alpha. subunits of the luciferases from B. harveyi and P. fischeri. The luciferase from B. harveyi is more sensitive to chymotrypsin than to trypsin; the luciferases of the Photobacterium spp. are more sensitive to trypsin than to chymotrypsin. Proteolytic inactivation of all 3 luciferases results from hydrolysis of a few peptide bonds in the .alpha. subunit; the proteolytic fragments from the 3 luciferases in 0.50 M phosphate are approximately the same size, indicating that the 3 enzymes have a protease-labile region at about the same position in the primary structure of their .alpha. subunits. Phosphate stabilizes all 3 luciferases against inactivation by proteases. Formation and degradation of intermediate species derived from the .alpha. subunits are readily observable in all 3 luciferases. Phosphate alters both the rate of product formation and the sites of peptide bond scission. The .beta. subunits of the luciferases from the 2 Photobacterium spp., unlike the enzyme of B. harveyi, appear to be degraded in buffers containing low concentrations of phosphate; in high-phosphate buffers, the .beta. subunits of all 3 luciferases appear to resist proteases. Analysis of native and chymotrypsin-inactivated P. fischeri and P. phosphoreum luciferases in the analytical ultracentrifuge indicates that, as with B. harveyi luciferase, the products of limited proteolysis do not dissociate under nondenaturing conditions. The fact that the luciferases from evolutionarily diverse species of luminous bacteria have protease-sensitive bonds in the same region of the .alpha. subunit that are stabilized by anions strongly suggests that the protease-labile region of the .alpha. subunit is either an integral component of or in close proximity to the active center.