Interaction of Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase with pyridoxal 5'-diphospho-5'-adenosine. Affinity labeling of Lys-21 and Lys-343.
Interaction of Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase with pyridoxal 5'-diphospho-5'-adenosine. Affinity labeling of Lys-21 and Lys-343.
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DOI：
10.1016/s0021-9258(19)67631-3
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发表时间：
1991-03
期刊：
影响因子：
--
通讯作者：
J. Ladine;D. Carlow;W. Lee;R. L. Cross;T. Flynn;H. Levy
中科院分区：
文献类型：
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作者：
J. Ladine;D. Carlow;W. Lee;R. L. Cross;T. Flynn;H. Levy
吡啶多醛5′-二磷酸-5′-腺苷（PLP-AMP）对葡萄糖-6-磷酸具有竞争性抑制作用，对NAD+或NADP+具有非竞争性抑制作用，在NADP-连锁反应中Ki = 40 μ m，在NADP-连锁反应中Ki = 34 μ m。葡萄糖-6-磷酸脱氢酶与[3H]PLP-AMP孵育，然后进行硼氢化物还原，结果表明，每mol酶亚基需要加入0.85 mol PLP-AMP才能完全失活。葡萄糖6-磷酸和NAD+都可以防止这种共价修饰。修饰酶的蛋白水解和标记肽的分离和测序表明，Lys-21和Lys-343是PLP-AMP相互作用的位点，葡萄糖6-磷酸和NAD+保护这两个赖氨酸残基免受修饰。吡哆醛5′-磷酸（PLP）也可以修饰Lys-21和Lys-343。Lys-21是高度保守区域的一部分，存在于所有已测序的葡萄糖-6-磷酸脱氢酶中。Lys-343与其他葡萄糖-6-磷酸脱氢酶中的精氨酸基残基相对应，并且与这些酶的同源性较低。PLP- amp和PLP被认为在葡萄糖-6-磷酸结合位点与肠系膜乳杆菌葡萄糖-6-磷酸脱氢酶相互作用。同时结合NAD+诱导构象变化(Kurlandsky, s.b, Hilburger, a.c, and Levy, h.r. (1988) Arch。物化学。生物物理学。264,93-102)，假设干扰希夫碱的形成与PLP或PLP- amp。PLP或PLP- amp共价修饰的赖氨酸残基中的一个或两个可能位于NAD（+）诱导构象变化的酶区域。
Pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP) inhibits glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides competitively with respect to glucose 6-phosphate and noncompetitively with respect to NAD+ or NADP+, with Ki = 40 microM in the NADP-linked and 34 microM in the NAD-linked reaction. Incubation of glucose-6-phosphate dehydrogenase with [3H]PLP-AMP followed by borohydride reduction shows that incorporation of 0.85 mol of PLP-AMP per mol of enzyme subunit is required for complete inactivation. Both glucose 6-phosphate and NAD+ protect against this covalent modification. The proteolysis of the modified enzyme and isolation and sequencing of the labeled peptides revealed that Lys-21 and Lys-343 are the sites of PLP-AMP interaction and that glucose 6-phosphate and NAD+ protect both lysyl residues against modification. Pyridoxal 5'-phosphate (PLP) also modifies Lys-21 and probably Lys-343. Lys-21 is part of a highly conserved region that is present in all glucose-6-phosphate dehydrogenases that have been sequenced. Lys-343 corresponds to an arginyl residue in other glucose-6-phosphate dehydrogenases and is in a region that is less homologous with those enzymes. PLP-AMP and PLP are believed to interact with L. mesenteroides glucose-6-phosphate dehydrogenase at the glucose 6-phosphate binding site. Simultaneous binding of NAD+ induces conformational changes (Kurlandsky, S. B., Hilburger, A. C., and Levy, H. R. (1988) Arch. Biochem. Biophys. 264, 93-102) that are postulated to interfere with Schiff's-base formation with PLP or PLP-AMP. One or both of the lysyl residues covalently modified by PLP or PLP-AMP may be located in regions of the enzyme undergoing the NAD(+)-induced conformational changes.