辣根过氧化物酶-氧化酶振荡器中的烟酰胺腺嘌呤二核苷酸种类

Nicotinamide adenine dinucleotide species in the horseradish peroxidase-oxidase oscillator.

作者信息

Kirkor E S, Scheeline A

机构信息

School of Chemical Sciences, University of Illinois at Urbana-Champaign, USA.

出版信息

Eur J Biochem. 2000 Aug;267(16):5014-22. doi: 10.1046/j.1432-1327.2000.01554.x.

DOI:10.1046/j.1432-1327.2000.01554.x

PMID:10931183

Abstract

NADH chemistry ancillary to the oscillatory peroxidase-oxidase (PO) reaction has been reexamined. Previously, (NAD)2 has been thought of as a terminal, inert product of the PO reaction. We now show that (NAD)2 is a central reactant in this system. Although we found traces of the dimer after several hours of the PO reaction, no accumulation of the dimer occurred, regardless of the reaction time or the number of oscillations. (NAD)2 can convert horseradish peroxidase (HRP) compound I (CpI) to compound II (CpII) with apparent rate constant (2.7 +/- 0.2) x 105 M-1.s-1 and CpII to HRP at 1 x 105 M-1.s-1. Moreover, a reduction of HRP compound III (CpIII) to CpI by (NAD)2 occurs with a rate constant faster than 5 x 106 M-1.s-1. The (NAD)2 reduction of CpIII provides an alternative to the reduction by NAD radical suggested by Yokota and Yamazaki. HRP catalyzes oxidation of alpha-NADH, not only the beta anomer as previously assumed. Rate constants of alpha- and beta-NADH reactions with CpI are (7.4 +/- 0.4) x 105 M-1.s-1, and (1.7 +/- 0.2) x 105 M-1.s-1, and with CpII are estimated as 5 x 104 M-1.s-1, and 4 x 104 M-1.s-1. Apparent rate constants of reduction of methylene blue (MB) to leuco-methylene blue (MBH) are 3.8 x 104 M-1.s-1 for NADH and 6.4 x 104 M-1.s-1 for NAD dimer, (NAD)2, while reoxidation of MBH proceeds at (2.1 +/- 0.2) x 103 M-1.s-1 All the rates were measured in 0.1 M acetate buffer, pH 5.1.

摘要

已对振荡过氧化物酶 - 氧化酶（PO）反应的辅助NADH化学过程进行了重新研究。此前，(NAD)₂被认为是PO反应的最终惰性产物。我们现在表明，(NAD)₂是该系统中的核心反应物。尽管在PO反应数小时后发现了痕量的二聚体，但无论反应时间或振荡次数如何，二聚体都不会积累。(NAD)₂可以将辣根过氧化物酶（HRP）化合物I（CpI）转化为化合物II（CpII），表观速率常数为(2.7±0.2)×10⁵ M⁻¹·s⁻¹，将CpII转化为HRP的速率为1×10⁵ M⁻¹·s⁻¹。此外，(NAD)₂将HRP化合物III（CpIII）还原为CpI的速率常数快于5×10⁶ M⁻¹·s⁻¹。(NAD)₂对CpIII的还原为横田和山崎提出的由NAD自由基进行的还原提供了一种替代方式。HRP催化α - NADH的氧化，而不仅仅是如先前假设的β异头物。α - 和β - NADH与CpI反应的速率常数分别为(7.4±0.4)×10⁵ M⁻¹·s⁻¹和(1.7±0.2)×10⁵ M⁻¹·s⁻¹，与CpII反应的速率估计分别为5×10⁴ M⁻¹·s⁻¹和4×10⁴ M⁻¹·s⁻¹。NADH将亚甲蓝（MB）还原为无色亚甲蓝（MBH）的表观速率常数为3.8×10⁴ M⁻¹·s⁻¹，NAD二聚体(NAD)₂为6.4×10⁴ M⁻¹·s⁻¹，而MBH的再氧化速率为(2.1±0.2)×10³ M⁻¹·s⁻¹。所有速率均在0.1 M醋酸盐缓冲液（pH 5.1）中测量。