Diethylpyrocarbonate inactivation of NAD-malic enzyme from Ascaris suum.
Diethylpyrocarbonate inactivation of NAD-malic enzyme from Ascaris suum.
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焦碳酸二乙酯灭活猪蛔虫中的 NAD-苹果酸酶。
DOI：
10.1016/0003-9861(85)90362-5
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发表时间：
1985
影响因子：
3.9
通讯作者：
Cook,PF
中科院分区：
文献类型：
--
作者：
Rao,JG;Harris,BG;Cook,PF
Abstract Treatment with diethylpyrocarbonate results in a first-order loss of the malate oxidative decarboxylase activity of NAD-malic enzyme. First-order plots are biphasic, with about 40–50% activity loss in the first phase. The inactivation process is not saturable, and the second-order rate constant is 4.7 spm− 1 s− 1 Malate (250 m m) provides complete protection against inactivation (as measured by a decrease in the inactivation rate), and less malate is required with Mg 2+ present. Partial protection (50%) is afforded by either NAD+(1 m m) or Mg 2+(50 m m). Treatment of modified (inactive) enzyme with hydroxylamine restores activity to 100% of the control when corrected for the effect of hydroxylamine on unmodified enzyme. A total of 10–13 histidine residues/subunit are acylated concomitant with loss of activity while 1–2 tyrosines are modified prior to any activity loss. The presence of Mg 2+ and malate at saturating concentrations protect 1–2 histidine residues/subunit. The intrinsic fluorescence of the enzyme decreases with time after addition of diethylpyrocarbonate, but the rate constant for this process is at least 10-fold too low to account for the biphasicity observed in the first order plots. The histidine modified which is responsible for loss of activity has ap K of 8.3 as determined from the pH dependence of the rate of inactivation. The histidine titrated is still modified under conditions where the residue is completely protonated but at a rate 1 100 the rate of the unprotonated histidine. The results suggest that 1–2 histidines are in or near the malate binding site and are required for malate oxidative decarboxylation.
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