Lysine residues involved in the hysteresis and in the regulatory sites of spinach ribulose 1,5-bisphosphate carboxylase/oxygenase.
Lysine residues have been suggested to be involved in the hysteretic decrease of the activity of spinach ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and the binding of ribulose 1,5-bisphosphate to its regulatory sites [Yokota, A. & Tsujimoto, N. (1992) Eur. J. Biochem. 204, 901-909]. This work identifies the lysine residues and investigates the effects of their chemical modification on the course of RuBisCO reaction. The carbamylated form of RuBisCO reacted with trinitrobenzene sulfonate in three phases; an initial rapid, second slow, and final non-specific reaction. Lys-334 in loop 6, Lys-21, and Lys-128, all from the large subunits, were trinitrophenylated in the first 60-min reaction. Lys-305 of the large subunits was labeled in the next step. The modification of these residues was strongly suppressed in the enzyme form that had undergone hysteretic conformational change after binding 2-carboxyarabinitol 1,5-bisphosphate at its catalytic sites. Instead, Lys-450 of the large subunits and Lys-71 from the small subunits were newly modified in the quaternary complex. A higher concentration of 2-carboxyarabinitol 1,5-bisphosphate reduced the trinitrophenylation of the two residues to half. The modification of the carbamylated form of the enzyme for 30 min was expected to arylate Lys-21, Lys-128, and Lys-334 at random, and the course of the reaction of the partially modified enzyme was expected to deviate from that of the unmodified enzyme. Experimental results showed that this was the case.(ABSTRACT TRUNCATED AT 250 WORDS)
Yokota
A
A
Department of Agricultural Chemistry, University of Osaka Prefecture.
Tokai
H
H
D000595:Amino Acid Sequence; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation; D008239:Lysine / Q000737:chemistry* / Q000378:metabolism; D008745:Methylation; D008969:Molecular Sequence Data; D010428:Pentosephosphates / Q000378:metabolism; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013402:Sugar Alcohols / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism; D014675:Vegetables / Q000201:enzymology*
D010428:Pentosephosphates; D013402:Sugar Alcohols; C032687:2-carboxyarabinitol 1,5-biphosphate; D014357:Trypsin; D012273:Ribulose-Bisphosphate Carboxylase; D008239:Lysine