硝基苯酯类与内酯类作为胞质醛脱氢酶底物的比较

A comparison of nitrophenyl esters and lactones as substrates of cytosolic aldehyde dehydrogenase.

Kitson T M, Kitson K E

Department of Chemistry and Biochemistry, Massey University, Palmerston North, New Zealand.

Biochem J. 1996 May 15;316 ( Pt 1)(Pt 1):225-32. doi: 10.1042/bj3160225.

Abstract

p-Nitrophenyl (PNP) acetate and propionate show a burst of p-nitrophenoxide release when their hydrolysis is catalysed by sheep liver cytosolic aldehyde dehydrogenase. This is not seen in the presence of NAD+ or NADH, implying a change in ratedetermining step. 2. 6-Nitrodihydrocoumarin (6-NDC) shows no burst of absorbance in the visible region. We propose that the pKa of the transient "reporter group' produced during the hydrolysis of this lactone is high (approx. 10) and that the incipient covalently linked p-nitrophenoxide moiety is protonated immediately on formation. The small burst seen in the hydrolysis of 5-nitro-2-coumaranone (5-NC) suggests that the pKa of its reporter group is about 8.5. 3. NADH markedly enhances the steady-state rate with the lactones. 5-NC shows a large rapid burst of colour development in the presence of NADH; this implies that NADH decreases the pKa of the reporter group to 7-7.5. 4. In the presence of NAD+, 5-NC and 6-NDC give an unusual "negative burst' in the stopped-flow traces. We propose that, under these circumstances, acylation of the enzyme is extremely fast and that the first event seen in the stopped-flow traces is protonation of the reporter group. NAD+ also greatly increases the steady-state rate. 5. With the lactones in the presence of NADH, the kcat value (nearly 6 s-1), a measure of the deacylation rate, is compatible with the single-site model for dehydrogenase and esterase activities.

摘要

对硝基苯乙酸酯（PNP）和对硝基苯丙酸酯在被羊肝细胞质醛脱氢酶催化水解时，会出现对硝基苯氧负离子的快速释放。在烟酰胺腺嘌呤二核苷酸（NAD⁺）或烟酰胺腺嘌呤二核苷酸磷酸（NADH）存在时则不会出现这种情况，这意味着限速步骤发生了变化。2. 6-硝基二氢香豆素（6-NDC）在可见光区域没有吸光度的快速变化。我们认为，该内酯水解过程中产生的瞬时“报告基团”的pKa值较高（约为10），并且初始的共价连接的对硝基苯氧负离子部分在形成后立即被质子化。5-硝基-2-香豆满酮（5-NC）水解时出现的小的快速变化表明其报告基团的pKa约为8.5。3. NADH显著提高了内酯的稳态水解速率。5-NC在NADH存在下会出现快速的大幅颜色变化；这意味着NADH将报告基团的pKa降低至7 - 7.5。4. 在NAD⁺存在下，5-NC和6-NDC在停流追踪中出现异常的“负快速变化”。我们认为，在这些情况下，酶的酰化极其迅速，并且在停流追踪中看到的第一个事件是报告基团的质子化。NAD⁺也大大提高了稳态速率。5. 对于在NADH存在下的内酯，催化常数（kcat）值（接近6 s⁻¹），即脱酰化速率的一种度量，与脱氢酶和酯酶活性的单位点模型相符。