Parker D M, Hardman M J, Plapp B V, Holbrook J J, Shore J D
Biochem J. 1978 Jul 1;173(1):269-75. doi: 10.1042/bj1730269.
Horse liver alcohol dehydrogenase specifically carboxymethylated on cysteine-46 (a ligand to the zinc in the active site) or acetimidylated on 25 of the 30 lysine residues per subunit (including residue 228) was studied. The tryptophan fluorescence of these enzymes decreased by 35% as pH was increased, with an apparent pKa of 9.8 +/- 0.2, identical with that of native enzyme. Native enzyme in the presence of 30mM-imidazole, which displaces a water molecule ligated to the zinc, also had a pKa of 9.8. The ionoizable group is thus neither the water molecule nor one of the modified groups. Binding of NAD+ shifted the pKa for the fluorescence transition to 7.6 with native enzyme and to 9.0 with acetimidylated enzyme, but did not shift the pKa of carboxymethylated enzyme. Binding of NAD+ and trifluoroethanol, an unreactive alcohol, gave maximal fluorescence quenching at pH7 with all three enzymes. The acetimidylated enzyme--NAD+--trifluoroethanol complex had an apparent pKa of 5.0, but the pK of the native enzyme complex was experimentally inaccessible. The results are interpreted in terms of coupled equilibria between two different conformational states. On binding of NAD+, the modified enzymes apparently change conformation less readily than does native enzyme, but binding of alcohol can drive the change to completion.
研究了在半胱氨酸-46（活性位点锌的配体）上特异性羧甲基化或在每个亚基30个赖氨酸残基中的25个（包括第228位残基）上乙酰亚胺化的马肝醇脱氢酶。随着pH值升高，这些酶的色氨酸荧光降低了35%，表观pKa为9.8±0.2，与天然酶相同。在30mM咪唑存在下的天然酶，该咪唑取代了与锌相连的水分子，其pKa也为9.8。因此，可电离基团既不是水分子也不是修饰基团之一。对于天然酶，NAD⁺的结合将荧光转变的pKa移至7.6，对于乙酰亚胺化酶则移至9.0，但未改变羧甲基化酶的pKa。NAD⁺和三氟乙醇（一种无反应性的醇）的结合在pH7时使所有三种酶的荧光猝灭达到最大值。乙酰亚胺化酶 - NAD⁺ - 三氟乙醇复合物的表观pKa为5.0，但天然酶复合物的pK在实验中无法测得。结果根据两种不同构象状态之间的耦合平衡进行解释。在结合NAD⁺时，修饰酶显然比天然酶更不容易改变构象，但醇的结合可以促使构象变化完成。
