Reversible dissociation and conformational stability of dimeric ribulose bisphosphate carboxylase.
Dimer-monomer dissociation of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum was investigated using hydrostatic pressure in the range 1-2 kbar to promote dissociation. Intrinsic fluorescence emission and polarization, along with the polarization of the fluorescence of single-labeled AEDANS conjugates, were used to follow the dissociation. Full reversibility after dissociation was observed to depend on the presence of small ligands: glycerol, Mg2+, and NaHCO3, the last two being required to activate the enzyme. The free energy of association at 15 degrees C, -12.9 kcal mol-1, was made up of a positive change in enthalpy on association of 6.0 kcal mol-1 and an entropic contribution (T delta S) of 18.9 kcal mol-1; thus the monomer association is entropy driven. No dissociation of the quaternary complex formed by the dimer, 2-carboxy-D-arabinitol 1,5-diphosphate (CADP), Mg2+, and NaHCO3 was observed at pressures up to 2.0 kbar; the magnitude of stabilization by the inhibitor binding was estimated as 2.3 kcal mol-1. Pressurization in the presence of bis-ANS results in a time-dependent increase in fluorophore emission, indicating changes in monomer conformation with exposure of hydrophobic surfaces upon dissociation. Reactivity against the fluorescent probe 1,5-I-AEDANS was also used as a conformational probe: HPLC of a trypsin digest of rubisco labeled at atmospheric pressure revealed a single fluorescent peptide, whereas more extensive labeling was observed when the reaction was carried out at 2.0 kbar, indicative of exposure of internal cysteines.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Biochemistry, University of Illinois, Urbana 61801.
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D001639:Bicarbonates / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D004795:Enzyme Stability; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D005990:Glycerol / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D006874:Hydrostatic Pressure; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009282:Naphthalenesulfonates; D011487:Protein Conformation; D012247:Rhodospirillum rubrum / Q000201:enzymology; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry / Q000378:metabolism*; D012964:Sodium / Q000494:pharmacology; D017693:Sodium Bicarbonate; D016339:Spectrometry, Mass, Fast Atom Bombardment; D013816:Thermodynamics
D001639:Bicarbonates; D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; C006421:1,5-I-AEDANS; D017693:Sodium Bicarbonate; D012964:Sodium; D012273:Ribulose-Bisphosphate Carboxylase; D008274:Magnesium; D005990:Glycerol