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 d ecrease of the activity of spinach ribulose 1,8-bisphosphate carboxyla se/oxygenase (RuBisCO) and the binding of ribulose 1,5-bisphosphate to its regulatory sites [Yokota, A. and Tsujimoto, N. (1992) Eur. J. Bio chem. 204, 901-909]. This work identifies the lysine residues and inve stigates the effects of their chemical modification on the course of R uBisCO reaction. The carbamylated form of RuBisCO reacted with trinitr obenzene 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-m in reaction. Lys-305 of the large subunits was labeled in the next ste p. The modification of these residues was strongly suppressed in the e nzyme form that had undergone hysteretic conformational change after b inding 2-carboxyarabinitol 1,5-bisphosphate at its catalytic sites. In stead, Lys-450 of the large subunits and Lys-71 from the small subunit s were newly modifed in the quaternary complex. A higher concentration of 2-carboxyarabinitol 1,5-bisphosphate reduced the trinitrophenylati on of the two residues to half. The modification of the carbamylated f orm 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 e nzyme. Experimental results showed that this was the case. Lys-21 may construct a salt bridge with Glu-53 of the same large subunit, and Lys -128 is close to the peptidic oxygens between Val-331 and Gly-333 of l oop 6 of the adjoining subunit in the large-subunit dimer in the quate rnary complex. We reason that the decrease in activity in the reaction course may be caused by the interactions of these lysine residues wit h other important residues.