Identification of residues in the aromatic substrate binding site of horseradish peroxidase by 1H NMR studies on isozymes.
Identification of residues in the aromatic substrate binding site of horseradish peroxidase by 1H NMR studies on isozymes.
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通过同工酶的 1 H NMR 研究鉴定辣根过氧化物酶芳香底物结合位点中的残基。
DOI：
10.1021/bi00041a027
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发表时间：
1995
期刊：
影响因子：
2.9
通讯作者：
Mar,GN
中科院分区：
文献类型：
--
作者：
deRopp,JS;Chen,Z;Mar,GN
两种辣根过氧化物酶酸性同工酶A1（HRPA 1，未测序）和A2（HRPA 2，测序）的氰化物抑制复合物已经通过溶液二维NMR方法检测，并且活性位点分子和电子结构与充分表征的同工酶C（HRPC）的活性位点分子和电子结构进行比较（Chen，Z.，de Ropp，J.S.，Hernández，G.，& La Mar，G. N.等人（1994）J. Am. 116，8772-8783），以及细胞色素c过氧化物酶。确定了HRPA 1-CN和HRPA 2-CN活性位点附近催化相关残基的身份和排列，并确定了酸性与C同工酶的差异催化特性中涉及的关键残基替换。血红素和轴向His接触移位模式，以及与血红素和彼此的残基的偶极接触，证实了三种同工酶之间的高度保守的结构，包括参与酶的活化的远端口袋残基。血红素口袋的显着的动态稳定性，反映在NH交换与溶剂，也是保守的三种同工酶。在HRPc-CN中鉴定了另外的血红素接触，lie 148。HRPc-CN中与血红素接触的四个残基在HRPA 2-CN中被取代，其中两个可能是功能中性的，Gly 169- 200。
The cyanide-inhibited complexes of two horseradish peroxidase acidic isozymes, Al (HRPA1, unsequenced) and A2 (HRPA2, sequenced), have been examined by solution two-dimensional NMR methods, and the active site molecular and electronic structure compared to that of the well-characterized isozyme C (HRPC)(Chen, Z., de Ropp, J. S., Hernández, G., & La Mar, G. N.(1994) J. Am. Chem. Soc. 116, 8772—8783), as well as to that of cytochrome c peroxidase. The identity and alignment of catalytically relevant residues near the active site for HRPA1-CN and HRPA2-CN are determined, and key residue replacements implicatedin the differential catalytic properties of the acidic vs C isozymes are identified. Heme and axial His contact shift patterns, as well as dipolar contacts of residues with the heme and with each other, confirm a highly conserved structure among the three isozymes, including for the distal pocket residues involved in the activation of the enzyme. The remarkable dynamic stability of the heme pocket, as reflected in NH exchange with solvent, is also conserved for the three isozymes. An additional heme contact, lie 148, is identified in HRPc-CN. Four residues in contact with the heme in HRPc-CN are replaced in HRPA2-CN, two of which are likely functionally neutral, Gly 169—