Robinson S P, Portis A R
U.S. Department of Agriculture, Agricultural Research Service and Department of Agronomy, University of Illinois, Urbana, Illinois 61801.
Plant Physiol. 1989 Jul;90(3):968-71. doi: 10.1104/pp.90.3.968.
The rate of CO(2) fixation by ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) following addition of ribulose 1,5-bisphosphate (RuBP) to fully activated enzyme, declined with first-order kinetics, resulting in 50% loss of rubisco activity after 10 to 12 minutes. This in vitro decline in rubisco activity, termed fall-over, was prevented if purified rubisco activase protein and ATP were added, allowing linear rates of CO(2) fixation for up to 20 minutes. Rubisco activase could also stimulate rubisco activity if added after fallover had occurred. Gel filtration of the RuBP-rubisco complex to remove unbound RuBP allowed full activation of the enzyme, but the inhibition of activated rubisco during fallover was only partially reversed by gel filtration. Addition of alkaline phosphatase completely restored rubisco activity following fallover. The results suggest that fallover is not caused by binding of RuBP to decarbamylated enzyme, but results from binding of a phosphorylated inhibitor to the active site of rubisco. The inhibitor may be a contaminant in preparations of RuBP or may be formed on the active site but is apparently removed from the enzyme in the presence of the rubisco activase protein.
向完全活化的酶中添加1,5 - 二磷酸核酮糖（RuBP）后，1,5 - 二磷酸核酮糖羧化酶/加氧酶（rubisco）固定CO₂的速率以一级动力学下降，导致10至12分钟后rubisco活性损失50%。如果添加纯化的rubisco活化酶蛋白和ATP，这种体外rubisco活性的下降（称为“fall - over”）可以被阻止，从而使CO₂固定速率在长达20分钟内保持线性。如果在“fall - over”发生后添加rubisco活化酶，也可以刺激rubisco活性。对RuBP - rubisco复合物进行凝胶过滤以去除未结合的RuBP可使酶完全活化，但凝胶过滤只能部分逆转“fall - over”期间活化rubisco的抑制作用。添加碱性磷酸酶可在“fall - over”后完全恢复rubisco活性。结果表明，“fall - over”不是由RuBP与脱氨甲酰化酶的结合引起的，而是由一种磷酸化抑制剂与rubisco活性位点的结合导致的。该抑制剂可能是RuBP制剂中的污染物，也可能在活性位点形成，但在rubisco活化酶蛋白存在的情况下显然会从酶中去除。
