Kasher J S, Allen K E, Kasamo K, Slayman C W
J Biol Chem. 1986 Aug 15;261(23):10808-13.
Treatment of the plasma membrane H+-ATPase of Neurospora crassa with the arginine-specific reagents phenylglyoxal or 2,3-butanedione at 30 degrees C, pH 7.0, leads to a marked inhibition of ATPase activity. MgATP, the physiological substrate of the enzyme, protects against inactivation. MgADP, a competitive inhibitor of ATPase activity with a measured Ki of 0.11 mM, also protects, yielding calculated KD values of 0.125 and 0.115 mM in the presence of phenylglyoxal and 2,3-butanedione, respectively. The excellent agreement between Ki and KD values makes it likely that MgADP exerts its protective effect by binding to the catalytic site of the enzyme. Loss of activity follows pseudo-first order kinetics with respect to phenylglyoxal and 2,3-butanedione concentration, and double log plots of pseudo-first order rate constants versus reagent concentration yield slopes of 0.999 (phenylglyoxal) and 0.885 (2,3-butanedione), suggesting that the modification of one reactive site/mol of H+-ATPase is sufficient for inactivation. This stoichiometry has been confirmed by direct measurements of the incorporation of [14C]phenylglyoxal. Taken together, the results support the notion that one arginine residue, either located at the catalytic site or shielded by a conformational change upon nucleotide binding, plays an essential role in Neurospora H+-ATPase activity.
在30℃、pH 7.0条件下，用精氨酸特异性试剂苯乙二醛或2,3-丁二酮处理粗糙脉孢菌的质膜H⁺-ATP酶，会导致ATP酶活性显著受到抑制。该酶的生理底物MgATP可防止其失活。MgADP是ATP酶活性的竞争性抑制剂，测得的Ki为0.11 mM，它也具有保护作用，在存在苯乙二醛和2,3-丁二酮时，计算得出的KD值分别为0.125 mM和0.115 mM。Ki和KD值之间的良好一致性表明MgADP可能通过与酶的催化位点结合来发挥其保护作用。失活遵循关于苯乙二醛和2,3-丁二酮浓度的假一级动力学，假一级速率常数与试剂浓度的双对数图给出的斜率分别为0.999（苯乙二醛）和0.885（2,3-丁二酮），这表明每摩尔H⁺-ATP酶中一个反应位点的修饰就足以导致失活。这种化学计量关系已通过对[¹⁴C]苯乙二醛掺入量的直接测量得到证实。综合来看，这些结果支持了这样一种观点，即一个精氨酸残基，要么位于催化位点，要么在核苷酸结合时因构象变化而被屏蔽，在粗糙脉孢菌H⁺-ATP酶活性中起着至关重要的作用。
