Mullins L S, Zawadzke L E, Walsh C T, Raushel F M
Department of Chemistry, Texas A & M University, College Station 77843.
J Biol Chem. 1990 Jun 5;265(16):8993-8.
The steady state kinetic mechanism, molecular isotope exchange and the positional isotope exchange (PIX) reactions of D-alanyl-D-alanine ligase from Salmonella typhimurium have been studied. The kinetic mechanism has been determined to be ordered Ter-Ter from initial velocity and product inhibition experiments. The first substrate to bind is ATP followed by the addition of 2 mol of D-alanine. Pi is released, and then D-alanyl-D-alanine and ADP dissociate from the enzyme surface. In the reverse direction D-alanyl-D-alanine exhibits complete substrate inhibition (Ki = 1.15 +/- 0.05 mM) by binding to the enzyme-ATP complex. In the presence of D-alanine, D-alanyl-D-alanine ligase catalyzed the positional exchange of the beta,gamma-bridge oxygen in [gamma-18O4]ATP to a beta-nonbridge position. Two possible alternate dead-end substrate analogs, D-2-chloropropionic acid and isobutyric acid, did not induce a positional isotope exchange in [gamma-18O4]ATP. The positional isotope exchange rate is diminished relative to the net substrate turnover as the concentration of D-alanine is increased. This is consistent with the ordered Ter-Ter mechanism as determined by the steady state kinetic experiments. The ratio of the positional isotope exchange rate relative to the net chemical turnover of substrate (Vex/Vchem) approaches a value of 1.4 as the concentration of D-alanine becomes very small. This ratio is 100 times larger than the ratio of the maximal reverse and forward chemical reaction velocities (V2/V1). This situation is only possible when the reaction mechanism proceeds in two distinct steps and the first step is much faster than the second step. The enzyme was also found to catalyze the molecular isotope exchange of radiolabeled D-alanine with D-alanyl-D-alanine in the presence of phosphate. These results are consistent with the formation of D-alanyl phosphate as a kinetically competent intermediate.
对鼠伤寒沙门氏菌D -丙氨酰 - D -丙氨酸连接酶的稳态动力学机制、分子同位素交换和位置同位素交换（PIX）反应进行了研究。通过初速度和产物抑制实验确定其动力学机制为有序的Ter - Ter机制。首先结合的底物是ATP，随后添加2摩尔的D -丙氨酸。释放出Pi，然后D -丙氨酰 - D -丙氨酸和ADP从酶表面解离。在逆向反应中，D -丙氨酰 - D -丙氨酸通过与酶 - ATP复合物结合表现出完全的底物抑制（Ki = 1.15±0.05 mM）。在D -丙氨酸存在下，D -丙氨酰 - D -丙氨酸连接酶催化[γ - 18O4]ATP中β,γ - 桥氧向β - 非桥位置的位置交换。两种可能的替代终产物类似物，D - 2 - 氯丙酸和异丁酸，未在[γ - 18O4]ATP中诱导位置同位素交换。随着D -丙氨酸浓度的增加，位置同位素交换速率相对于底物的净周转量降低。这与稳态动力学实验确定的有序Ter - Ter机制一致。当D -丙氨酸浓度变得非常小时，位置同位素交换速率相对于底物净化学周转的比率（Vex/Vchem）接近1.4。该比率比最大逆向和正向化学反应速度的比率（V2/V1）大100倍。只有当反应机制分两个不同步骤进行且第一步比第二步快得多时，这种情况才可能发生。还发现该酶在磷酸盐存在下催化放射性标记的D -丙氨酸与D -丙氨酰 - D -丙氨酸的分子同位素交换。这些结果与形成动力学上可行的中间体D -丙氨酰磷酸一致。
