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)
Erijman
L
L
Department of Biochemistry, University of Illinois, Urbana 61801.
Lorimer
G H
GH
Weber
G
G
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