Skipper L, Campbell W H, Mertens J A, Lowe D J
Biological Chemistry Department, John Innes Centre, Norwich NR4 7UH, United Kingdom.
J Biol Chem. 2001 Jul 20;276(29):26995-7002. doi: 10.1074/jbc.M100356200. Epub 2001 May 16.
Recombinant Arabidopsis NADH:nitrate reductase was expressed in Pichia pastoris using fermentation. Large enzyme quantities were purified for pre-steady-state kinetic analysis, which had not been done before with any eukaryotic nitrate reductase. Basic biochemical properties of recombinant nitrate reductase were similar to natural enzyme forms. Molybdenum content was lower than expected, which was compensated for by activity calculation on molybdenum basis. Stopped-flow rapid-scan spectrophotometry showed that the enzyme FAD and heme were rapidly reduced by NADH with and without nitrate present. NADPH reduced FAD at less than one-tenth of NADH rate. Reaction of NADH-reduced enzyme with nitrate yielded rapid initial oxidation of heme with slower oxidation of flavin. Rapid-reaction freeze-quench EPR spectra revealed molybdenum was maintained in a partially reduced state during turnover. Rapid-reaction chemical quench for quantifying nitrite production showed that the rate of nitrate reduction was initially greater than the steady-state rate, but rapidly decreased to near steady-state turnover rate. However, rates of internal electron transfer and nitrate reduction were similar in magnitude with no one step in the catalytic process appearing to be much slower than the others. This leads to the conclusion that the catalytic rate is determined by a combination of rates with no overall rate-limiting individual process.
利用发酵技术在毕赤酵母中表达了重组拟南芥NADH：硝酸还原酶。纯化了大量该酶用于稳态前动力学分析，此前尚未对任何真核生物硝酸还原酶进行过此类分析。重组硝酸还原酶的基本生化特性与天然酶形式相似。钼含量低于预期，这通过基于钼的活性计算得到了弥补。停流快速扫描分光光度法表明，无论有无硝酸盐存在，该酶的黄素腺嘌呤二核苷酸（FAD）和血红素都能被NADH迅速还原。NADPH还原FAD的速率不到NADH的十分之一。NADH还原的酶与硝酸盐反应，导致血红素迅速发生初始氧化，黄素氧化较慢。快速反应冷冻淬灭电子顺磁共振光谱显示，在周转过程中钼保持在部分还原状态。用于定量亚硝酸盐产生的快速反应化学淬灭表明，硝酸盐还原速率最初大于稳态速率，但迅速下降至接近稳态周转速率。然而，内部电子传递速率和硝酸盐还原速率在数量上相似，催化过程中没有一个步骤明显比其他步骤慢得多。由此得出结论，催化速率是由多种速率共同决定的，不存在整体限速的单个过程。
