Properties of rat 6-N-trimethyl-L-lysine hydroxylases: similarities among the kidney, liver, heart, and skeletal muscle activities.
Properties of rat 6-N-trimethyl-L-lysine hydroxylases: similarities among the kidney, liver, heart, and skeletal muscle activities.
复制标题
大鼠 6-N-三甲基-L-赖氨酸羟化酶的特性：肾、肝、心脏和骨骼肌活动之间的相似性。
DOI：
10.1016/0003-9861(82)90508-2
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发表时间：
1982
影响因子：
3.9
通讯作者：
Englard,S
中科院分区：
文献类型：
--
作者：
Stein,R;Englard,S
已检查并比较了几种大鼠组织将 6-N-三甲基-L-赖氨酸 (lys(Me3)) 羟基化为 3-羟基-6-N-三甲基-L-赖氨酸 (3-HO-lys(Me3)) 的情况。此前研究表明，肾酶需要分子氧和 α-酮戊二酸作为辅助底物，亚铁和抗坏血酸作为辅助因子，并受到过氧化氢酶的刺激，在 37°C 时具有广泛的最适 pH 值，范围为 6.5 至 7.5。根据肾、肝、心脏和骨骼肌的粗组织提取物测定，底物、辅底物和辅因子获得了相似的表观 Km 值。鉴于在大鼠组织中检测的几种lys(Me3)羟化酶之间的相似动力学参数，以及骨骼肌lys(Me3)羟化酶活性水平与心脏、肝脏和肾脏的活性水平相当的事实，因为其总质量较大，骨骼肌可能对lys(Me3)生物合成l-肉碱有显着贡献。与 lys(Me3) 竞争的最有效抑制剂是 2-N-乙酰基-6-N-三甲基-L-赖氨酸、6-N-单甲基-L-赖氨酸和 6-N-二甲基-L-赖氨酸。l-2-氨基-6-N-三甲基铵-4-己烯酸，d-2-氨基-6-N-三甲基铵-4-己烯酸，和dl2-氨基-6-N-三甲基铵-顺-4-己烯酸也抑制羟化酶活性，但机制尚未确定。草乙酸盐、琥珀酸盐和柠檬酸盐通过与α-酮戊二酸竞争来抑制羟基化反应。二价铁与酶的结合受到“软金属”离子（例如 Cd2+、Zn2+）的竞争性抑制，但不受“硬金属”离子（例如 Ca2+、Mg2+）的竞争性抑制。将粗肾酶与苯磺酸汞、N-乙基马来酰亚胺、碘乙酸盐或碘乙酰胺在 37°C 下预孵育 15 分钟，可显着抑制 3-HO-lys(Me3) 的形成。通过在酶预孵育过程中加入 Zn (II)，可以降低 N-乙基马来酰亚胺的抑制程度。 “软”金属和巯基试剂对酶的影响表明，活性位点的亚铁结合需要巯基。
Hydroxylation of 6-N-trimethyl-l-lysine(lys(Me3)) to 3-hydroxy-6-N-trimethyl-l-lysine(3-HO-lys(Me3)) by several rat tissues has been examined and compared. The kidney enzyme, which previously was shown to require molecular oxygen and α-ketoglutarate as cosubstrates, ferrous iron and ascorbate as cofactors, and to be stimulated by catalase, has a broad pH optimum ranging between 6.5 to 7.5 at 37 °C. As determined with crude tissue extracts from kidney, liver, heart, and skeletal muscle, similar apparentKmvalues were obtained for substrate, cosubstrates, and cofactors. In view of similar kinetic parameters among the several lys(Me3) hydroxylases examined in rat tissues, and the fact that the level of skeletal muscle lys(Me3) hydroxylase activity is comparable to that of heart, liver, and kidney, because of its large total mass, skeletal muscle may contribute significantly to the biosynthesis ofl-carnitine from lys(Me3). The most effective inhibitors found, competitive with lys(Me3), were 2-N-acetyl-6-N-trimethyl-l-lysine, 6-N-monomethyl-l-lysine, and 6-N-dimethyl-l-lysine.l-2-Amino-6-N-trimethylammonium-4-hexynoate,d-2-amino-6-N-trimethylammonium-4-hexynoate, anddl2-amino-6-N-trimethylammonium-cis-4-hexenoate, also inhibited hydroxylase activity but by a yet undetermined mechanism. Oxalacetate, succinate, and citrate inhibited the hydroxylation reaction by competing with α-ketoglutarate. The binding of ferrous iron to the enzyme was competitively inhibited by ions of “soft metals” (e.g., Cd2+, Zn2+) but not by those of “hard metals” (e.g., Ca2+, Mg2+). Preincubation of the crude kidney enzyme for 15 min at 37 °C with mercuriphenylsulfonate,N-ethylmaleimide, iodoacetate, or iodoacetamide resulted in considerable inhibition of 3-HO-lys(Me3) formation. The degree of inhibition byN-ethylmaleimide could be reduced by including Zn (II) during preincubation of the enzyme. The effects of “soft” metals and sulfhydryl reagents on the enzyme suggest that sulfhydryl groups are required for ferrous iron binding in the active site.