Robinson J D
Department of Pharmacology, SUNY Health Science Center, Syracuse 13210.
Biochim Biophys Acta. 1989 Feb 2;994(2):95-103. doi: 10.1016/0167-4838(89)90148-9.
(Na+ + K+)-ATPase activity of a dog kidney enzyme preparation was markedly inhibited by 10-30% (v/v) dimethyl sulfoxide (Me2SO) and ethylene glycol (Et(OH)2); moreover, Me2SO produced a pattern of uncompetitive inhibition toward ATP. However, K+-nitrophenylphosphatase activity was stimulated by 10-20% Me2SO and Et(OH)2 but was inhibited by 30-50%. Me2SO decreased the Km for this substrate but had little effect on the Vmax below 30% (at which concentration Vmax was then reduced). Me2SO also reduced the Ki for Pi and acetyl phosphate as competitors toward nitrophenyl phosphate but increased the Ki for ATP, CTP and 2-O-methylfluorescein phosphate as competitors. Me2SO inhibited K+-acetylphosphatase activity, although it also reduced the Km for that substrate. Finally, Me2SO increased the rate of enzyme inactivation by fluoride and beryllium. These observations are interpreted in terms of the E1P to E2P transition of the reaction sequence being associated with an increased hydrophobicity of the active site, and of Me2SO mimicking such effects by decreasing water activity: (i) primarily to stabilize the covalent E2P intermediate, through differential solvation of reactants and products, and thereby inhibiting the (Na+ + K+)-ATPase reaction and acting as a dead-end inhibitor to produce the pattern of uncompetitive inhibition; inhibiting the K+-acetylphosphatase reaction that also passes through an E2P intermediate; but not inhibiting (at lower Me2SO concentrations) the K+-nitrophenylphosphatase reaction that does not pass through such an intermediate; and (ii) secondarily to favor partitioning of Pi and non-nucleotide phosphates into the hydrophobic active site, thereby decreasing the Km for nitrophenyl phosphate and acetyl phosphate, the Ki for Pi and acetyl phosphate in the K+-nitrophenylphosphatase reaction, accelerating inactivation by fluoride and beryllium acting as phosphate analogs, and, at higher concentrations, inhibiting the K+-nitrophenylphosphatase reaction by stabilizing the non-covalent E2.P intermediate of that reaction. In addition, Me2SO may decrease binding at the adenine pocket of the low-affinity substrate site, represented as an increased Ki for ATP, CTP and 3-O-methylfluorescein phosphate.
犬肾酶制剂的（钠+钾）-ATP酶活性受到10 - 30%（体积/体积）二甲基亚砜（Me2SO）和乙二醇（Et(OH)2）的显著抑制；此外，Me2SO对ATP产生非竞争性抑制模式。然而，钾-对硝基苯磷酸酶活性受到10 - 20% Me2SO和Et(OH)2的刺激，但受到30 - 50%的抑制。Me2SO降低了该底物的米氏常数（Km），但在低于30%（在此浓度下最大反应速度（Vmax）随后降低）时对Vmax影响很小。Me2SO还降低了磷酸根离子（Pi）和乙酰磷酸作为对硝基苯磷酸竞争物的抑制常数（Ki），但增加了ATP、三磷酸胞苷（CTP）和2 - O - 甲基荧光素磷酸作为竞争物的Ki。Me2SO抑制钾-乙酰磷酸酶活性，尽管它也降低了该底物的Km。最后，Me2SO增加了氟化物和铍对酶的失活速率。这些观察结果可根据反应序列从E1P到E2P的转变与活性位点疏水性增加相关来解释，以及Me2SO通过降低水活性模拟这种效应来解释：（i）主要通过反应物和产物的差异溶剂化来稳定共价E2P中间体，从而抑制（钠+钾）-ATP酶反应并作为终产物抑制剂产生非竞争性抑制模式；抑制同样经过E2P中间体的钾-乙酰磷酸酶反应；但在较低Me2SO浓度下不抑制不经过此类中间体的钾-对硝基苯磷酸酶反应；（ii）其次有利于Pi和非核苷酸磷酸分配到疏水性活性位点，从而降低对硝基苯磷酸和乙酰磷酸的Km、钾-对硝基苯磷酸酶反应中Pi和乙酰磷酸的Ki，加速作为磷酸类似物的氟化物和铍的失活作用，并且在较高浓度下通过稳定该反应的非共价E2.P中间体来抑制钾-对硝基苯磷酸酶反应。此外，Me2SO可能降低在低亲和力底物位点腺嘌呤口袋处的结合，表现为ATP、CTP和3 - O - 甲基荧光素磷酸的Ki增加。
