Thomas O. Baldwin

The effects of phosphate on the structure and stability of the luciferases from Beneckea, harveyi, Photobacterium, fischeri, and, Photobacterium phosphoreum

Abstract The luciferases from three very distinct species of luminous bacteria, Beneckea , harveyi , Photobacterium , fischeri , and , Photobacterium , phosphoreum , bind phosphate and other anions to form complexes with decreased sensitivity to proteases and increased thermal stability. Upon treatment with either trypsin or chymotrypsin in either low phosphate or high phosphate buffers, the rate of loss of activity of all three luciferases is the same as the rate of bond cleavage within a discrete region of the α subunit. Unlike the enzyme from B. , harveyi , the β subunits of the enzymes from the Photobacterium species are sensitive to the proteases in low phosphate, while in high phosphate, the β subunits of all three luciferases are not sensitive to trypsin or chymotrypsin.

Inactivation of luciferase from the luminous marine bacterium Beneckea harveyi by proteases: Evidence for a protease labile region and properties of the protein following inactivation

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.

Modification of the reactive sulfhydryl of bacterial luciferase with spin-labeled maleimides☆

Abstract Luciferase from the luminous marine bacterium Beneckea harveyi has been modified at a single cysteinyl residue by reaction with an equimolar amount of a spin-labeled maleimide: 3-(maleimidomethyl)-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl (I) or 3-(3-maleimidopropyl carbamoyl)-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl (II). The resulting enzymes modified with compound I or with compound II were inactive. The apparent second-order rate constants for reaction of luciferase with I and II at pH 7.0, 25 °C, were (5.7 ± 0.1) × 104 and (1.1 ± 0.2) × 105 m −1 min−1, respectively. Examination of the labeled luciferases by electron spin resonance spectroscopy indicated that the polarity of the region occupied by the nitroxide, and thus presumably of the active center, is similar to that of 2-propanol and that the reactive sulfhydryl resides in a cleft at least 17 A in length.

Studies on the complex formed between bacitracin A and divalent cations.

Bacitracin A is a peptide antibiotic which forms stoichiometric complexes with divalent cations, including Ni2+ and Zn2+. In this paper it is shown that the metal-bacitracin complex contains a group which has a pKa near pH 5.5. Deprotonation of the group is concomitant with the aggregation and precipitation of the metal-bacitracin complex. Bacitracin A, in the absence of metals, does not contain any group which has a pKa in this range. It is postulated that this group is the N-terminal amino of isoleucine, which was previously postulated not to be directly involved in metal coordination based on proton release measurements. An attempt was made to demonstrate directly that the N-terminal amino group is not coordinated to the metal by examining the reactivity of this group with 2,4,6-trinitrobenzene sulfonate. It was clearly shown that bound metals protect the N-terminal amino group from reacting with this reagent. It is speculated that this metal-protection results from a combination of factors, including steric hindrance.