Diversa reports financial results for 2Q and 1H ended Jun 2002

Abstract

We have previously reported (T. O. Baldwin, J. W. Hastings, and P. L. Riley, 1978, J. Biol. Chem., 253, 5551–5554) that the proteolytic inactivation of the luciferase from the luminous marine bacterium Beneckea harveyi is due to hydrolysis of one or a small number of peptide bonds within the α subunit, and that following proteolytic inactivation, the molecular weight of the protein measured by sedimentation equilibrium is unaltered. We have continued these investigations and have made the following observations: (1) The proteaselabile region of the α subunit is about 20–25 residues in length, and located approximately 100–125 residues from one of the termini of the subunit; it possesses five to six trypsin-sensitive sites and two chymotrypsin-sensitive sites. (2) No binding could be measured between either the substrate, reduced flavin mononucleotide, or the product, oxidized flavin mononucleotide, and the luciferase inactivated with trypsin. (3) The results of sedimentation velocity measurements (Holzman, T. F., and Baldwin, T. O., 1980, Proc. Nat. Acad. Sci. USA, in press), reaction of the protein thiols with 5,5′-dithiobis(2-nitrobenzoic acid), and ultraviolet circular dichroism measurements suggest that following proteolytic inactivation, the luciferase has a slightly more expanded structure. (4) Analysis of a mutant luciferase, AK-6, having an α-subunit lesion that results in a red-shifted bioluminescence emission spectrum and a greatly reduced affinity for reduced flavin mononucleotide shows that alterations in the active center can result in a dramatically altered sensitivity to proteases, both in the rate of inactivation and in the sites of peptide bond hydrolysis.