Effects of mutations in M4 of the gastric H+,K+-ATPase on inhibition kinetics of SCH28080.
Effects of mutations in M4 of the gastric H+,K+-ATPase on inhibition kinetics of SCH28080.
复制标题
胃 H ,K -ATP 酶 M4 突变对 SCH28080 抑制动力学的影响。
作者：
Munson,KB;Lambrecht,N;Sachs,G
用定点突变的方法研究了M4残基对胃H+，K+-ATPase选择性K+竞争性抑制剂SCH28080表观KI的影响。描述了一种利用HEK293细胞和编码H+，K+-ATP酶α和β亚基的单独质粒的双转染表达系统。该野生型酶的比活力(每毫克表达的H+，K+-ATPase蛋白的微摩尔数为Piper小时)，对氨(K+的替代物)明显Km，对SCH28080的明显Ki值相当于从猪胃粘膜中纯化的H+，K+-ATPase。α亚基M4跨膜区的氨基酸是从对SCH28080不敏感的Na+，K+-ATPase的M4区非保守残基中挑选出来的。大多数突变产生的活性酶具有相似的Km，APP值为NH4+和KI，APP值为SCH28080。SCH28080在M330V中亲和力降低2倍，在M334I和V337I中亲和力降低9倍，而对Km、APP无明显影响。因此，蛋氨酸334和缬氨酸337参与结合，但不是NH4+结合部位的一部分。甲硫氨酸可能位于抑制部位的外围，抑制部位的最小尺寸必须为∼16×8×5ä，并且可以从E2-P构象的管腔进入。多个序列比对将膜表面放置在精氨酸328附近，这表明M4螺旋一侧的蛋氨酸334和缬氨酸337的侧链突出到膜结构域内的结合腔中。
The effects of site-directed mutagenesis were used to explore the role of residues in M4 on the apparentKiof a selective, K+-competitive inhibitor of the gastric H+,K+ATPase, SCH28080. A double transfection expression system is described, utilizing HEK293 cells and separate plasmids encoding the α and β subunits of the H+,K+-ATPase. The wild-type enzyme gave specific activity (micromoles of Piper hour per milligram of expressed H+,K+-ATPase protein), apparentKmfor ammonium (a K+surrogate), and apparentKifor SCH28080 equal to the H+,K+-ATPase purified from hog gastric mucosa. Amino acids in the M4 transmembrane segment of the α subunit were selected from, and substituted with, the nonconserved residues in M4 of the Na+,K+-ATPase, which is insensitive to SCH28080. Most of the mutations produced competent enzyme with similarKm,appvalues for NH4+andKi,appfor SCH28080. SCH28080 affinity was decreased 2-fold in M330V and 9-fold in both M334I and V337I without significant effect onKm,app. Hence methionine 334 and valine 337 participate in binding but are not part of the NH4+site. Methionine 330 may be at the periphery of the inhibitor site, which must have minimum dimensions of ∼16 × 8 × 5 Å and be accessible from the lumen in the E2-P conformation. Multiple sequence alignments place the membrane surface near arginine 328, suggesting that the side chains of methionine 334 and valine 337, on one side of the M4 helix, project into a binding cavity within the membrane domain.