Mutation of the conserved domains of two inositol polyphosphate 5-phosphatases
Mutation of the conserved domains of two inositol polyphosphate 5-phosphatases
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DOI：
10.1021/bi9602627
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发表时间：
1996-06-18
期刊：
影响因子：
2.9
通讯作者：
Majerus, PW
中科院分区：
文献类型：
--
作者：
Jefferson, AB;Majerus, PW
两个短的氨基酸基序WXGDXNXR和PXWCDRXL定义了肌醇多聚5-磷酸酶的一个大家族。我们通过在血小板75 kDa多聚磷酸肌醇5-磷酸酶II(5-磷酸酶II)中将这些保守氨基酸中的每一个突变为丙氨酸来测试这些保守氨基酸对底物结合和催化的重要性。在杆状病毒感染的Sf9细胞中表达了天然形式和突变形式的5-磷酸酶II，并用Mono-Q柱层析纯化了重组蛋白，并对其活性进行了研究。突变体D476A、N478A、D553A和R554A在所有四种已知的底物上都没有检测到该酶的活性。突变株R480A、W551A和I555A对INS(1，4，5)P-3的水解率显著低于天然酶[K-m=75mU M，V-m=8300nmolINS(1，4，5)P-3水解酶min(-1)(mg蛋白质)(-1)]。突变株W551A和I555A对INS(1，4，5)P-3的Km与天然酶相似(分别为35mM和81mM)，表明这些氨基酸不起底物结合作用。相反，突变体R480A既增加了K-m(634µM)，又降低了V-m[INS(1，4，5)P-3水解分(-1)(蛋白毫克)(-1)]。通过K-m测定，突变体R480A保留了INS(1，3，4，5)P-4的正常结合，表明基序2中的精氨酸在INS(1，4，5)P-3结合中的作用大于INS(1，3，4，5)P-4的结合。突变体I555A与Ins(1，3，4，5)P-4结合亲和力降低8倍。这些突变显著降低了另外三种底物Ins(1，3，4，5)P-4，PtdIns(4，5)P-2和PtdIns(3，4，5)P-3的5-磷酸酶II的水解率。我们还测试了一个与D553A，D460A类似的突变，在110 kDa的信号转导肌醇多聚磷酸5-磷酸酶(5SIP110)中。5SIP110 D460A没有检测到酶活性，但保留了与Grb2结合的能力。这些结果与这些保守氨基酸在底物结合和催化中的作用是一致的。
Two short amino acid motifs, WXGDXNXR and PXWCDRXL, define a large family of inositol polyphosphate 5-phosphatases. We tested the importance of seven of these conserved amino acids to substrate binding and catalysis by mutating each to alanine in the platelet 75 kDa inositol polyphosphate 5-phosphatase II (5-phosphatase II). Native and mutant forms of 5-phosphatase II were expressed in baculovirus-infected Sf9 cells, and the recombinant proteins were purified by Mono Q chromatography and studied for enzyme activity. Mutants D476A, N478A, D553A, and R554A had no detectable activity using all four known substrates for this enzyme. Mutants R480A, W551A, and I555A showed greatly reduced hydrolysis of Ins(1,4,5)P-3 when compared to native enzyme [K-m = 75 mu M, V-m = 8300 nmol of Ins(1,4,5)P-3 hydrolyzed min(-1) (mg of protein)(-1)]. Mutants W551A and I555A had a K-m for Ins(1,4,5)P-3 hydrolysis similar to that of the native enzyme (35 mu M and 81 mu M, respectively), suggesting that these amino acids do not play a role in binding substrate. By contrast, mutant R480A had both increased K-m (634 mu M) and decreased V-m [855 nmol of Ins(1,4,5)P-3 hydrolyzed min(-1) (mg of protein)(-1)]. As judged by measurement of K-m, mutant R480A retained normal binding of Ins(1,3,4,5)P-4, suggesting that the arginine in motif 2 has a greater role in Ins(1,4,5)P-3 binding than in Ins(1,3,4,5)P-4 binding. Mutant I555A bound Ins(1,3,4,5)P-4 with 8-fold reduced affinity. These mutations markedly reduced 5-phosphatase II hydrolysis of the three other substrates, Ins(1,3,4,5)P-4, PtdIns(4,5)P-2, and PtdIns(3,4,5)P-3. We also tested a mutation comparable to D553A, D460A, in the 110 kDa form of the signaling inositol polyphosphate 5-phosphatase (5SIP110). 5SIP110 D460A had no detectable enzyme activity but retained the ability to bind GRB2. These results are consistent with a role for these conserved amino acids in substrate binding and catalysis.