Jaworowski A, Rose I A
J Biol Chem. 1985 Jan 25;260(2):944-8.
When the enzymatically generated intermediate 2-carboxy-3-keto-D-arabinitol-1,5-bisphosphate (II) was used as a substrate with fresh enzyme, 70% reacted to produce 3-phosphoglycerate (3PGA). When a reaction mixture of enzyme plus [1-32P]ribulose 1,5-bisphosphate (RuBP) was quenched in the steady state with the tightly bound inhibitor 2-carboxyarabinitol-1,5-bisphosphate, 30% of the enzyme-bound species was released as 3PGA and 70% as RuBP. The major source for this partition was the ternary substrates Michaelis complex. The level of carboxylated intermediate in the steady state was determined to be 8% of active sites under the conditions of substrate saturation. No burst was seen in the appearance of product when 6.5 eq of [1-32P]RuBP was mixed with enzyme plus saturating CO2 and the reaction followed in the steady state. From these data plus the steady-state Vmax and Km of RuBP it is possible to derive the five bulk rate constants represented in the scheme ECO2 + RuBP in equilibrium ERuBPCO2 in equilibrium E X II----E + 2(3PGA).
当将酶促生成的中间体2-羧基-3-酮-D-阿拉伯糖醇-1,5-二磷酸（II）作为新鲜酶的底物时，70%发生反应生成3-磷酸甘油酸（3PGA）。当酶加[1-32P]核酮糖1,5-二磷酸（RuBP）的反应混合物在稳态下用紧密结合的抑制剂2-羧基阿拉伯糖醇-1,5-二磷酸淬灭时，30%的酶结合物以3PGA形式释放，70%以RuBP形式释放。这种分配的主要来源是三元底物米氏复合物。在底物饱和条件下，稳态下羧化中间体的水平被确定为活性位点的8%。当6.5当量的[1-32P]RuBP与酶加饱和CO2混合并在稳态下跟踪反应时，产物出现时没有观察到爆发。根据这些数据以及RuBP的稳态Vmax和Km，可以推导出方案ECO2 + RuBP⇌ERuBPCO2⇌E X II→E + 2(3PGA)中表示的五个总体速率常数。
