Essential tryptophan residues of ribulose 1,5-bisphosphate carboxylase.
Ribulose 1,5-bisphosphate carboxylase [3-phospho-D-glyceratecarboxy-lyase (dimerizing), EC 4.1.1.39] is rapidly and irreversibly inactivated by micromolar concentrations of dimethyl (2-hydroxy-5-nitrobenzyl) sulphonium bromide (DMHNB), a tryptophan selective reagent, after reversible protection of the reactive sulphydryl groups. The inactivation followed pseudo-first-order reaction kinetics. Replots of the kinetic data indicated that no reversible enzyme-inhibitor complex was formed prior to irreversible modification. Kinetic analysis and the correlation of the spectral data at 410 nm with enzyme activity indicated that inactivation by DMHNB resulted from modification of on an average one tryptophan per 67 kDa combination of large and small subunits. Several competitive inhibitors and substrate RuBP offered strong protection against inhibition. The k1/2 (protection) for RuBP was 1.3 mM, indicating that the tryptophan residues may be located at or near the substrate binding site. Free and total sulphydryl groups were not affected by the reagent. The modified enzyme exhibited significantly reduced intrinsic fluorescence, indicating that the microenvironment of the tryptophans at the active site is significantly perturbed. Tryptic peptide profiles and CD spectral analyses suggested that inactivation may not be due to the extensive conformational changes in the enzyme molecule during modification.
Purohit
U S
US
Molecular Biology and Agriculture Division, Bhabha Atomic Research Centre, Trombay, Bombay, India.
Bhagwat
A S
AS
D001665:Binding Sites; D012273:Ribulose-Bisphosphate Carboxylase / Q000032:analysis* / Q000037:antagonists & inhibitors; D013452:Sulfonium Compounds / Q000494:pharmacology; D014364:Tryptophan / Q000032:analysis*
D013452:Sulfonium Compounds; C024586:dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium; D014364:Tryptophan; D012273:Ribulose-Bisphosphate Carboxylase