de Ropp J S, Chen Z, Mar G N
Department of Chemistry, University of California, Davis 95616, USA.
Biochemistry. 1995 Oct 17;34(41):13477-84. doi: 10.1021/bi00041a027.
The cyanide-inhibited complexes of two horseradish peroxidase acidic isozymes, A1 (HRPA1, unsequenced) and A2 (HRPA2, sequenced), have been examined by solution two-dimensional 1H 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 implicated in 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, Ile 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-->Ala and Ile 148-->Leu. However, two substitutions in the acidic isozymes in the aromatic substrate binding pocket on the heme edge, Ile 244-->Leu and Phe 179 or 221-->aliphatic residue, could well account for the dramatic decrease (approximately 10(3) in aromatic substrate binding in the A1 and A2 isozymes vs the C isozyme of HRP.(ABSTRACT TRUNCATED AT 250 WORDS)
已通过溶液二维¹H NMR方法研究了两种辣根过氧化物酶酸性同工酶A1（HRPA1，未测序）和A2（HRPA2，已测序）的氰化物抑制复合物，并将活性位点的分子和电子结构与已充分表征的同工酶C（HRPC）（Chen, Z., de Ropp, J.S., Hernández, G., & La Mar, G.N. (1994) J. Am. Chem. Soc. 116, 8772 - 8783）以及细胞色素c过氧化物酶的进行了比较。确定了HRPA1 - CN和HRPA2 - CN活性位点附近催化相关残基的身份和排列，并鉴定了与酸性同工酶和C同工酶催化特性差异相关的关键残基替换。血红素和轴向组氨酸的接触位移模式，以及残基与血红素之间以及彼此之间的偶极接触，证实了这三种同工酶之间具有高度保守的结构，包括参与酶激活的远端口袋残基。血红素口袋显著的动态稳定性，如通过与溶剂的NH交换所反映的，在这三种同工酶中也得以保留。在HRPC - CN中鉴定出一个额外的血红素接触位点，即Ile 148。在HRPA2 - CN中，与HRPC - CN中接触血红素的四个残基被替换，其中两个可能在功能上是中性的，即Gly 169→Ala和Ile 148→Leu。然而，酸性同工酶中血红素边缘芳香底物结合口袋中的两个替换，Ile 244→Leu和Phe 179或221→脂肪族残基，很可能解释了A1和A2同工酶与HRP的C同工酶相比芳香底物结合显著降低（约10³）的原因。（摘要截短于250字）
