ABSTRACT
The propeptides of the vitamin K dependent blood clotting and regulatory proteins contain a gamma-carboxylation recognition site that directs precursor forms of these proteins for posttranslational gamma-carboxylation. Peptides corresponding to the propeptide of prothrombin were synthesized and examined by circular dichroism (CD) and nuclear magnetic resonance spectroscopy (NMR). CD spectra indicate that these peptides have little or no secondary structure in aqueous solutions but that the addition of trifluoroethanol induces or stabilizes a structure containing alpha-helical character. The maximum helical content occurs at 35-40% trifluoroethanol. This trifluoroethanol-stabilized structure was solved by two-dimensional NMR spectroscopy. The NMR results demonstrate that residues -13 to -3 form an amphipathic alpha-helix. NMR spectra indicate that a similar structure is present at 5 degrees C, in the absence of trifluoroethanol. Of the residues previously implicated in defining the gamma-carboxylation recognition site, four residues (-18, -17, -16, and -15) are adjacent to the helical region and one residue (-10) is located within the helix. The potential role of the amphipathic alpha-helix in the gamma-carboxylation recognition site is discussed.
Subject(s)
Carbon-Carbon Ligases , Ligases/chemistry , Protein Precursors/chemistry , Prothrombin/chemistry , Amino Acid Sequence , Binding Sites , Circular Dichroism , Cold Temperature , Enzyme Stability/drug effects , Magnetic Resonance Spectroscopy , Molecular Sequence Data , Peptide Fragments/chemical synthesis , Protein Conformation , Trifluoroethanol/pharmacologyABSTRACT
The luminescence of the CO adduct of two isozymic tyrosinases isolated from Agaricus bispora, an edible white mushroom, has been studied. At room temperature the emission appears as a single smooth peak centered at 530 nm with FWHM of 2700 cm-1 and a lifetime of 36 microseconds. The lifetime and wavelength of the emission are virtually unchanged on lowering the temperature from 298 to 77 degrees K. Solvent composition affects the wavelength of emission minimally. The emission is quenched by oxygen but not by a series of substrate analogs, inhibitors, or Lewis bases. The emission further appeared independent of aggregation state of the enzyme or isozyme type. A comparison of these data is made with those obtained by other researchers for the tyrosinase from Neurospora crassa and for several hemocyanins. The comparison supports the hypothesis that regulation of enzymatic activity does not take place within the coordination sphere of the copper atom observed. In addition, it suggests that the 550- to 560-nm emissions previously observed may not be considered characteristic of all CO derivatives of coupled binuclear copper proteins.
