Bilin Specificity for Thioether Linkage Formation

The question of bilin chromophore precursor specificity for holophytochrome assembly has been addressed using zinc blot analysis, difference spectroscopy, and fluorescence spectroscopy (15,37,40). The requirements for assembly are an A-ring ethylidene at the C3 position, as is present in POB, PCB, and PEB (37), and a C10 methine bridge. The former conclusion is based on in vivo feeding of BVs IIIa, IXa, and XIIIa; BV IXa and XIIIa feeding restored levels of spectrally active phy-tochrome, while BV IIIa had no apparent effect (15). Assembly of a bilin with an eth-ylidene at the C2 position cannot be ruled out, but this compound is not readily available, and based on BV IIIa feeding experiments, it is probably not biologically relevant. The requirement for a C10 methine bridge is based on the inability of rubins, including those possessing an A-ring eth-ylidene moiety, such as phycocyanorubin, to assemble with apophytochrome (61). The observations that the D-ring can be modified including 18-vinyl reduction,

Scheme 2. Reversible competitive inhibition of PEB phytochrome assembly. A kinetic model for PEB adduct formation in the presence of a reversible competitive inhibitor such as BV. The kinetics of PEB adduct formation should be pseudo-first-order as predicted by Equation 7. The raw fluorescence data is transformed and replotted as described by Equation 8. The slopes of these semilog replots yield kjpp values. These values are used to construct the plot described by Equation 10. The x-intercept of Equation 10 yields an estimate of the equilibrium dissociation constant for the reversible competitive inhibitor.

Scheme 3. Irreversible inhibition of PEB phytochrome assembly. A kinetic model for PEB adduct formation in the presence of an irreversible competitive inhibitor such as POB. The formation of both PEB-phytochrome and competitor bilin—phytochrome are described by Equations 11 and 12. In the presence of large molar excesses of all bilins, these equations are first-order expressions. The ^app1 values are calculated using Equation 13, then plotted as a function of the competitive inhibitor according to Equations 14 and 15. A plot of Equation 15 yields the dissociation constant (K) and the catalytic rate constant (k^) for the competitive inhibitor of fluorescent adduct formation.

switching of the C17 and C18 methyl and vinyl moieties and elaboration of the C18 side chain reveal that the bilin binding pocket of apophytochrome is not very discerning with regard to the C18 substituent (15,37,40). The C15 methine bridge is not required for assembly, as demonstrated by the binding of PEB to apophytochome. BVs and bilirubins, which lack the A-ring ethylidene moiety, also do not form cova-lent adducts with phytochrome, although the former are capable of noncovalent

Figure 3. Fluorescence assay for holophytochrome assembly. Representative data for standard fluorescence analysis of PEB attachment to recombinant oat phytochrome A, after Li et al. (39). The upper panel shows raw fluorescence kinetic data as a function of increasing PEB concentration. The middle panel is a replot of the same data according to Equation 3 (Scheme 1), from which kapp values were estimated. The bottom panel depicts a replot of 1/kapp versus 1/(PEB) according to Equation 5 (Scheme 1). See text and Reference 39 for details.

Figure 3. Fluorescence assay for holophytochrome assembly. Representative data for standard fluorescence analysis of PEB attachment to recombinant oat phytochrome A, after Li et al. (39). The upper panel shows raw fluorescence kinetic data as a function of increasing PEB concentration. The middle panel is a replot of the same data according to Equation 3 (Scheme 1), from which kapp values were estimated. The bottom panel depicts a replot of 1/kapp versus 1/(PEB) according to Equation 5 (Scheme 1). See text and Reference 39 for details.

interaction with phytochrome and, therefore, can act as reversible competitive inhibitors as discussed above. The requirement for both propionic acid moieties has been established by the inability of bilin— esters to bind to apophytochrome (3).

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