Frillingos S, Gonzalez A, Kaback H R
Howard Hughes Medical Institute, Department of Physiology, University of California-Los Angeles 90095-1662, USA.
Biochemistry. 1997 Nov 25;36(47):14284-90. doi: 10.1021/bi972314d.
Cys-scanning mutagenesis has been applied to the remaining 45 residues in lactose permease that have not been mutagenized previously (from Gln100 to Arg144 which comprise helix IV and adjoining loops). Of the 45 single-Cys mutants, 26 accumulate lactose to > 75% of the steady state observed with Cys-less permease, and 14 mutants exhibit lower but significant levels of accumulation (35-65% of Cys-less permease). Permease with Phe140-->Cys or Lys131-->Cys exhibits low activity (15-20% of Cys-less permease), while mutants Gly115-->Cys, Glu126-->Cys and Arg144-->Cys are completely unable to accumulate the dissacharide. However, Cys-less permease with Ala or Pro in place of Gly115 is highly active, and replacement of Lys131 or Phe140 with Cys in wild-type permease has a less deleterious effect on activity. In contrast, mutant Glu126-->Cys or Arg144-->Cys is inactive with respect to both uphill and downhill transport in either Cys-less or wild-type permease. Furthermore, mutants Glu126-->Ala or Gln and Arg144-->Ala or Gln are also inactive in both backgrounds, and activity is not rescued by double neutral replacements or inversion of the charged residues at these positions. Finally, a mutant with Lys in place of Arg144 accumulates lactose to about 25% of the steady state of wild-type, but at a slow rate. Replacement of Glu126 with Asp, in contrast, has relatively little effect on activity. None of the effects can be attributed to decreased expression of the mutants, as judged by immunoblot analysis. Although the activity of most of the single-Cys mutants is unaffected by N-ethylmaleimide, Cys replacement at three positions (Ala127, Val132, or Phe138) renders the permease highly sensitive to alkylation. The results indicate that the cytoplasmic loop between helices IV and V, where insertional mutagenesis has little effect on activity [McKenna, E., et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 11954-11958], contains residues that play an important role in permease activity and that a carboxyl group at position 126 and a positive charge at position 144 are absolutely required.
半胱氨酸扫描诱变已应用于乳糖通透酶中先前未诱变的其余45个残基（从Gln100到Arg144，它们构成螺旋IV和相邻的环）。在45个单半胱氨酸突变体中，26个积累乳糖的量达到无半胱氨酸通透酶稳态的75%以上，14个突变体积累量较低但仍有显著水平（为无半胱氨酸通透酶的35 - 65%）。具有Phe140→Cys或Lys131→Cys的通透酶活性较低（为无半胱氨酸通透酶的15 - 20%），而突变体Gly115→Cys、Glu126→Cys和Arg144→Cys完全无法积累双糖。然而，用Ala或Pro取代Gly115的无半胱氨酸通透酶活性很高，在野生型通透酶中用Cys取代Lys131或Phe140对活性的有害影响较小。相比之下，突变体Glu126→Cys或Arg144→Cys在无半胱氨酸或野生型通透酶中对向上和向下运输均无活性。此外，突变体Glu126→Ala或Gln以及Arg144→Ala或Gln在两种背景下也均无活性，并且在这些位置的双中性取代或带电残基的反转均无法恢复活性。最后，用Lys取代Arg144的突变体积累乳糖的量约为野生型稳态的25%，但速率较慢。相比之下，用Asp取代Glu126对活性的影响相对较小。通过免疫印迹分析判断，这些影响均不能归因于突变体表达的降低。尽管大多数单半胱氨酸突变体的活性不受N - 乙基马来酰亚胺的影响，但在三个位置（Ala127、Val132或Phe138）进行半胱氨酸取代会使通透酶对烷基化高度敏感。结果表明，螺旋IV和V之间的胞质环，插入诱变对其活性影响较小[麦肯纳，E.等人（1992年）美国国家科学院院刊89，11954 - 11958]，其中包含在通透酶活性中起重要作用的残基，并且126位的羧基和144位的正电荷是绝对必需的。
