Nishimura J S, Mitchell T
J Biol Chem. 1984 Feb 25;259(4):2144-8.
The hypothesis that Escherichia coli succinyl-CoA synthetase functions by a cooperative alternating sites mechanism is based largely on the results of [18O]phosphate exchange experiments (Bild, G. S., Janson, C. A., and Boyer, P. D. (1980) J. Biol. Chem. 255, 8109-8115). In those experiments, [18O]Pi----succinate (predominantly) exchange appeared to proceed at greater rates (relative to the apparent amount of succinyl-CoA released from the enzyme) at low ATP in incubations containing ATP, CoA, succinate, [18O]Pi, 0.48 M hydroxylamine (as a succinyl-CoA trap), and a pyruvate kinase-lactate dehydrogenase ADP trap. The conclusion arrived at was that succinyl-CoA binding at one site was inversely related to ATP binding at the second site. Thus, the residence time of succinyl-CoA binding at a site would be longer at lower ATP concentrations. Our experiments show that, under the incubation conditions described by Bild et al. (Bild, G. S., Janson, C. A., and Boyer, P. D. (1980) J. Biol. Chem. 255, 8109-8115), succinyl-CoA is not efficiently trapped. Thus, at ATP concentrations from 3.6 to 150 microM, concentrations of succinyl-CoA from 13 to 78 microM were observed. Succinate----succinyl-CoA exchange reactions carried out in this range of ATP and subsaturating succinyl-CoA concentrations were found to be markedly inhibited by the addition of the ADP trap. This inhibition was more pronounced at higher ATP levels. At a saturating succinyl-CoA concentration (1.5 mM), addition of the ADP trap actually stimulated succinate----succinyl-CoA exchange. Under these conditions, ATP----Pi exchange was greatly depressed. These results are interpreted as follows. ADP is required for optimal binding of succinyl-CoA, but only when the latter is present at subsaturating concentrations; thus, the ADP trap inhibits the reaction. ATP exerts its stimulatory action on succinate---- succinyl-CoA exchange through an "other site" effect, i.e. in binding to the noncatalytic site of succinyl-CoA synthetase, it facilitates binding and release of succinyl-CoA at the catalytic site. ATP may also exert negative effects by inhibiting other site binding of ATP or by interfering with same site succinyl-CoA binding at subsaturating concentrations of the latter. These data support the notion that a half-sites mechanism applies to succinyl-CoA synthetase, but suggest that the [18O]Pi----succinate exchange data which have been instrumental in development of the cooperative alternating sites hypothesis should be re-evaluated.
大肠杆菌琥珀酰辅酶A合成酶通过协同交替位点机制发挥作用这一假说，很大程度上是基于[18O]磷酸盐交换实验的结果（比尔德，G. S.，詹森，C. A.，以及博耶，P. D.（1980年）《生物化学杂志》255卷，8109 - 8115页）。在那些实验中，在含有ATP、辅酶A、琥珀酸、[18O]Pi、0.48 M羟胺（作为琥珀酰辅酶A捕获剂）以及丙酮酸激酶 - 乳酸脱氢酶ADP捕获剂的孵育体系中，[18O]Pi→琥珀酸（主要是）交换似乎在低ATP浓度下（相对于从酶中释放出的琥珀酰辅酶A的表观量）以更高的速率进行。得出的结论是，一个位点上琥珀酰辅酶A的结合与第二个位点上ATP的结合呈负相关。因此，在较低的ATP浓度下，琥珀酰辅酶A在位点上的停留时间会更长。我们的实验表明，在比尔德等人所描述的孵育条件下（比尔德，G. S.，詹森，C. A.，以及博耶，P. D.（1980年）《生物化学杂志》255卷，8109 - 8115页），琥珀酰辅酶A没有被有效地捕获。因此，在ATP浓度为3.6至150微摩尔时，观察到琥珀酰辅酶A的浓度为13至78微摩尔。发现在这个ATP范围和亚饱和琥珀酰辅酶A浓度下进行的琥珀酸→琥珀酰辅酶A交换反应，因添加ADP捕获剂而受到显著抑制。在较高的ATP水平下，这种抑制更为明显。在饱和的琥珀酰辅酶A浓度（1.5毫摩尔）下，添加ADP捕获剂实际上刺激了琥珀酸→琥珀酰辅酶A交换。在这些条件下，ATP→Pi交换被极大地抑制。这些结果的解释如下。ADP是琥珀酰辅酶A最佳结合所必需的，但仅当后者以亚饱和浓度存在时；因此，ADP捕获剂抑制了反应。ATP通过“其他位点”效应，即通过与琥珀酰辅酶A合成酶的非催化位点结合，对琥珀酸→琥珀酰辅酶A交换发挥其刺激作用，它促进了催化位点上琥珀酰辅酶A的结合和释放。ATP也可能通过抑制ATP的其他位点结合或在琥珀酰辅酶A亚饱和浓度下干扰同一位点的琥珀酰辅酶A结合而产生负面影响。这些数据支持了半位点机制适用于琥珀酰辅酶A合成酶这一观点，但表明有助于协同交替位点假说发展的[18O]Pi→琥珀酸交换数据应该重新评估。
