Differential Activity and Structure of Highly Similar Peroxidases. Spectroscopic, Crystallographic, and Enzymatic Analyses of Lignifying Arabidopsis thaliana Peroxidase A2 and Horseradish Perox
详细信息 查看全文
- 作者：Kå ; re Lehmann Nielsen ; Chiara Indiani ; Anette Henriksen ; Alessandro Feis ; Maurizio Becucci ; Michael Gajhede ; Giulietta Smulevich ; and Karen G. Welinder
- 刊名：Biochemistry
- 出版年：2001
- 出版时间：September 18, 2001
- 年：2001
- 卷：40
- 期：37
- 页码：11013 - 11021
- 全文大小：269K
- 年卷期：v.40,no.37(September 18, 2001)
- ISSN：1520-4995
文摘
Anionic Arabidopsis thaliana peroxidase ATP A2 was expressed in Escherichia coli and usedas a model for the 95% identical commercially available horseradish peroxidase HRP A2. The crystalstructure of ATP A2 at 1.45 Å resolution at 100 K showed a water molecule only 2.1 Å from heme iron[Østergaard, L., et al. (2000) Plant Mol. Biol. 44, 231-243], whereas spectroscopic studies of HRP A2in solution at room temperature [Feis, A., et al. (1998) J. Raman Spectrosc. 29, 933-938] showed five-coordinated heme iron, which is common in peroxidases. Presented here, the X-ray crystallographic, single-crystal, and solution resonance Raman studies at room temperature confirmed that the sixth coordinationposition of heme iron of ATP A2 is essentially vacant. Furthermore, electronic absorption and resonanceRaman spectroscopy showed that the heme environments of recombinant ATP A2 and glycosylated plantHRP A2 are indistinguishable at neutral and alkaline pH, from room temperature to 12 K, and are highlyflexible compared with other plant peroxidases. Østergaard et al. (2000) also demonstrated that ATP A2expression and lignin formation coincide in Arabidopsis tissues, and docking of lignin precursors into thesubstrate binding site of ATP A2 predicted that coniferyl and p-coumaryl alcohols were good substrates.In contrast, the additional methoxy group of the sinapyl moiety gave rise to steric hindrance, not only inA2 type peroxidases but also in all peroxidases. We confirm these predictions for ATP A2, HRP A2, andHRP C. The specific activity of ATP A2 was lower than that of HRP A2 (pH 4-8), although a steady-state study at pH 5 demonstrated very little difference in their rate constants for reaction with H2O2 (k1= 1.0 
M-1 s-1). The oxidation of coniferyl alcohol, ferulic, p-coumaric, and sinapic acids by HRP A2,and ATP A2, however, gave modest but significantly different k3 rate constants of 8.7 ± 0.3, 4.0 ± 0.2,0.70 ± 0.03, and 0.04 ± 0.2 M-1 s-1 for HRP A2, respectively, and 4.6 ± 0.2, 2.3 ± 0.1, 0.25 ± 0.01,and 0.01 ± 0.004 M-1 s-1 for ATP A2, respectively. The structural origin of the differential reactivityis discussed in relation to glycosylation and amino acid substitutions. The results are of general importanceto the use of homologous models and structure determination at low temperatures.
M-1 s-1). The oxidation of coniferyl alcohol, ferulic, p-coumaric, and sinapic acids by HRP A2,and ATP A2, however, gave modest but significantly different k3 rate constants of 8.7 ± 0.3, 4.0 ± 0.2,0.70 ± 0.03, and 0.04 ± 0.2 M-1 s-1 for HRP A2, respectively, and 4.6 ± 0.2, 2.3 ± 0.1, 0.25 ± 0.01,and 0.01 ± 0.004 M-1 s-1 for ATP A2, respectively. The structural origin of the differential reactivityis discussed in relation to glycosylation and amino acid substitutions. The results are of general importanceto the use of homologous models and structure determination at low temperatures.