Wood P G, Müller H, Sovak M, Passow H
Max-Planck-Institut für Biophysik, Frankfurt/Main, Federal Republic of Germany.
J Membr Biol. 1992 Apr;127(2):139-48. doi: 10.1007/BF00233286.
The effect of mutation of either Lys 558 or Lys 869 or both on mouse erythroid band 3 protein (AE1)-mediated 36Cl- efflux and its inhibition by pyridoxal 5-phosphate (P5-P), DNDS and H2DIDS were studied. Regardless of the mutation, band 3 was always capable of executing Cl- self-exchange. P5-P (5 mM, pH 7.6) produced irreversible inhibition in the wild type (KK) and in the mutant in which Lys 558 (NK) or Lys 869 (KM) had been replaced by asparagine (N) or methionine (M), respectively. However, when both residues were replaced, mutant (NM), irreversible inhibition could no longer be achieved. This shows that P5-P is capable of producing inhibition with either one of the lysine residues, 558 or 869. Inhibition by DNDS changed dramatically upon mutation. The Ki app increased from 6.0 microM in the wild type (KK) to 23 microM in the mutant NK, to 73 microM in the mutant KM and to 474 microM in the double mutant NM. The Km value for activation of the transport system by varying the substrate concentration by isosmotic substitution of Cl- with SO4(2-) decreased from 42 mM in the wild type (KK) to 11.3 mM in the mutant NM. The results show that both Lys 558 and Lys 869 are involved in the maintenance of the structure of the overlapping binding sites for stilbene disulfonates and the substrate Cl-. In the double mutant NM, H2DIDS is no longer able to produce irreversible inhibition at pH 7.6. This is evidently related to the replacement of Lys 558 (pK 8.2) by Asn 558 in this mutant (see Bartel, D., Lepke, S., Layh-Schmitt, G., Legrum, B., Passow, H., 1989. EMBO J. 8:3601-3609). However, at pH 9.5, some irreversible inhibition could still be observed. This suggests that the other lysine residue (pK 10.8) that is known to be involved in covalent binding with the second isothiocyanate group of H2DIDS is still present, and hence, not identical to Lys 869, which had been substituted by a methionine residue. However, this result remains inconclusive since after mutagenesis, the H2DIDS may produce inhibition at a site that is not normally involved in H2DIDS binding.
研究了赖氨酸558或赖氨酸869或两者同时突变对小鼠红细胞带3蛋白（AE1）介导的36Cl-外流的影响，以及磷酸吡哆醛（P5-P）、DNDS和H2DIDS对其的抑制作用。无论是否发生突变，带3蛋白始终能够进行Cl-自我交换。P5-P（5 mM，pH 7.6）对野生型（KK）以及赖氨酸558（NK）或赖氨酸869（KM）分别被天冬酰胺（N）或甲硫氨酸（M）取代的突变体产生不可逆抑制。然而，当两个残基都被取代时，即突变体（NM），则不再能实现不可逆抑制。这表明P5-P能够通过赖氨酸残基558或869中的任何一个产生抑制作用。DNDS的抑制作用在突变后发生了显著变化。野生型（KK）的表观抑制常数Ki从6.0 microM增加到突变体NK中的23 microM，再到突变体KM中的73 microM，以及双突变体NM中的474 microM。通过用SO4(2-)等渗替代Cl-来改变底物浓度从而激活转运系统的米氏常数Km值，从野生型（KK）中的42 mM降至双突变体NM中的11.3 mM。结果表明，赖氨酸558和赖氨酸869都参与了芪二磺酸盐和底物Cl-重叠结合位点结构的维持。在双突变体NM中，H2DIDS在pH 7.6时不再能够产生不可逆抑制。这显然与该突变体中赖氨酸558（pK 8.2）被天冬酰胺558取代有关（见Bartel, D., Lepke, S., Layh-Schmitt, G., Legrum, B., Passow, H., 1989. EMBO J. 8:3601 - 3609）。然而，在pH 9.5时，仍可观察到一些不可逆抑制。这表明已知参与与H2DIDS的第二个异硫氰酸酯基团共价结合的另一个赖氨酸残基（pK 10.8）仍然存在，因此，它与已被甲硫氨酸残基取代的赖氨酸869不同。然而，由于诱变后，H2DIDS可能在一个通常不参与H2DIDS结合的位点产生抑制作用，所以这个结果仍然没有定论。
