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

AUTOR(ES)

Holzman, T F

RESUMO

Upon limited proteolysis of luciferases from the luminous marine bacteria Photobacterium fischeri, Photobacterium phosphoreum, and Beneckea harveyi, the rate of loss of luciferase activity is the same as the rate of loss of the heavier subunit of all three 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 species are more sensitive to trypsin than to chymotrypsin. Proteolytic inactivation of all three luciferases results from hydrolysis of a few peptide bonds in the alpha subunit; the proteolytic fragments from the three luciferases in 0.50 M phosphate are approximately the same size, indicating that the three enzymes have a protease-labile region at about the same position in the primary structure of their alpha subunits. Phosphate stabilizes all three luciferases against inactivation by proteases. Formation and degradation of intermediate species derived from the alpha subunits are readily observable in all three luciferases. Phosphate alters both the rate of product formation and the sites of peptide bond scission. The beta subunits of the luciferases from the two Photobacterium species, 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 three 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.

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