Binding of Phosphorylated Effectors by Active and Inactive Forms of Ribulose-1, 5-bisphosphate Carboxylase

Activation of ribulose1,5-bisphosphate carboxylase by CO, and Mgz+ is slow and reversible. At subsaturating concentrations of CO, and Mg2+, positive effectors increase and negative effectors decrease the amount of active enzyme at equilibrium. Preequilibrium experiments indicated that both positive and negative effectors inhibit the rates of enzyme activation and deactivation. Greater than 99% inhibition of the activation and deactivation rates was observed at high effector concentrations, indicating that the binding and release of the activators COz and Mg2+ occur only with effector-free enzyme. The deactivation rate Ki values for the negative effector ribose 5-phosphate and the positive effectors inorganic phosphate, fructose 1,6-bisphosphate, and 6-phosphogluconate were smaller than the corresponding activation rate Ki values by factors of 2, 6, 25, and 670, respectively. Thus, phosphorylated effectors impede deactivation more than activation. Equilibrium binding studies indicated that the active and inR i b u l o s e 1,5-bisphosphate carboxylase (EC 4.1.1.39) requires the addition of C 0 2 and a divalent metal ion in forming an activated enzyme complex capable of catalyzing the carboxylation and oxygenation of RuBP1 (Lorimer et al., 1976). The kinetics of this process have been well characterized for several RuBPCase enzymes purified from microbial and plant sources (Badger, 1980; Christeller & Laing, 1978; Laing & Christeller, 1976). The rate-limiting step in C02/Me2+ activation is carbamate formation at a lysine residue (Lys-201) on the 56-kDa catalytic subunit of the enzyme (Lorimer, 1981; Lorimer & Miziorko, 1980), and this is followed by rapid From the Department of Agricultural Biochemistry, University of Nebraska-Lincoln, Lincoln, Nebraska 68583-0718 (D.B.J. and R.C.), the Agronomy Department, University of Illinois, Urbana, Illinois 61 801 (D.B.J. and W.L.O.), and the U S . Department of Agriculture, Agricultural Research Service, Urbana, Illinois 61801 (W.L.O.). Received December 22, 1982. Research sponsored in part by Grant 78-59-231 10-1-1 19-1 from the USDA Competitive Research Grants Office (R.C.). Published as Paper No. 7046, Journal Series, Nebraska Agricultural Experiment Station. 0006-2960/83 10422-34 lO$O 1.5010 active enzyme forms have similar affinities for the positive effectors inorganic phosphate (KD = 650 pM) and fructose bisphosphate (KD = 11 pM). The positive effector 3phosphoglycerate was bound with greater affinity by the inactive enzyme (KD = 25 pM) than by the active enzyme (KD = 76 MM). Thus, preferential binding of positive effectors to the active enzyme form is not responsible for the enhancement of enzyme activation at equilibrium. The promotion of activation by positive effectors is mediated by altering the relative rates of activation and deactivation to favor active enzyme. Equilibrium binding studies indicated that the inactive enzyme had a much greater affinity for ribose 5-phosphate (KD = 42 pM) than did the active enzyme (KD = 480 pM). Preferential binding of this negative effector to inactive enzyme exceeds its stabilizing effect on the active enzyme form and thus causes an overall reduction of activation at equilibrium. addition of the metal cofactor as shown in eq 1. E E-C02 E-CO2-Me2+ (1) inactive active Although the mechanisms of activation and inhibition of RuBPCase by sugar phosphates and other anions have received considerable attention, these processes are not well resolved. Because of their potential regulatory role in vivo, chloroplast metabolites such as 6-P-gluconate, FBP, inorganic phosphate, and NADPH have been of primary interest. Several sugar phosphates enhance the activation state of RuBPCase which is partially activated with respect to C 0 2 and Mg2+, while other sugar phosphates lower the activation state of the enzyme ~ ~~~~~~~ ~ ~~~~~ ~~~~~ ~~ ~~ ' Abbreviations: RuBP, ribulose 1,5-bisphosphate; RuBPCase, ribulose1,5-bisphosphate carboxylase; CABP, 2-carboxyarabinitol 1,5-bisphosphate; FBP, fructose 1,6-bisphosphate; Bicine, N,N-bis(2-hydroxyethy1)glycine; P, phosphate; kDa, kilodalton; EDTA, ethylenediaminetetraacetic acid.