De Sandro V, Dupuy C, Kaniewski J, Ohayon R, Dème D, Virion A, Pommier J
Rhône-Poulenc Rorer, Département Sécurité du Médicament, Vitry sur Seine, France.
Eur J Biochem. 1991 Oct 15;201(2):507-13. doi: 10.1111/j.1432-1033.1991.tb16310.x.
The mechanism of NADPH oxidation catalyzed by horse-radish peroxidase (HRP) and 2,4-diacetyl-[2H]heme-substituted horse-radish peroxidase (DHRP) was studied. The roles of the different H2O2/peroxidase compounds were examined by spectral studies. The oxidized NADPH species were identified using the superoxide dismutase effect and by measuring the stoichiometry between NADPH oxidized and H2O2 used. In the presence of a mediating molecule, like scopoletin, both enzymes acted via a similar mechanism, producing only NADP degrees, which in turn reacted with O2 producing O2-. Consequently H2O2 was completely regenerated in the presence of superoxide dismutase and partially regenerated in its absence. In the absence of a mediating molecule, the H2O2 complex of both enzymes (compound I) catalysed NADPH oxidation by single-electron transfer, producing NADP degrees; compound II of these enzymes catalyzed NADPH oxidation more slowly by a direct two-electron transfer, producing NADPH+. There were difference between HRP and DHRP. HRP compound II was produced by the oxidation of 1 mol NADPH/mole compound I, while DHRP compound II was formed by the spontaneous conversion of compound I to compound II. The NADPH oxidation catalyzed by DHRP compound I did not lead to the formation of compound II. When H2O2 was produced slowly by the glucose/glucose-oxidase system, compound II was never formed and a pure O2- adduct of DHRP (compound III) accumulated.
研究了辣根过氧化物酶（HRP）和2,4-二乙酰基-[2H]血红素取代的辣根过氧化物酶（DHRP）催化NADPH氧化的机制。通过光谱研究考察了不同H2O2/过氧化物酶化合物的作用。利用超氧化物歧化酶效应并通过测量氧化的NADPH与消耗的H2O2之间的化学计量关系来鉴定氧化的NADPH种类。在存在介体分子（如 scopoletin）的情况下，两种酶通过类似的机制起作用，仅产生NADP +，其继而与O2反应生成O2-。因此，在存在超氧化物歧化酶的情况下H2O2完全再生，而在其不存在时部分再生。在不存在介体分子的情况下，两种酶的H2O2复合物（化合物I）通过单电子转移催化NADPH氧化，产生NADP +；这些酶的化合物II通过直接双电子转移催化NADPH氧化的速度较慢，产生NADPH +。HRP和DHRP之间存在差异。HRP化合物II由1摩尔NADPH/摩尔化合物I氧化产生，而DHRP化合物II由化合物I自发转化为化合物II形成。DHRP化合物I催化的NADPH氧化不会导致化合物II的形成。当通过葡萄糖/葡萄糖氧化酶系统缓慢产生H2O2时，从未形成化合物II，并且DHRP的纯O2-加合物（化合物III）积累。
