Malhotra O P, Bernhard S A
Proc Natl Acad Sci U S A. 1973 Jul;70(7):2077-81. doi: 10.1073/pnas.70.7.2077.
The absorption spectrum of an activesite specific chromophoric acyl enzyme, sturgeon beta-(2-furyl)-acryloyl-glyceraldehyde-3-phosphate dehydrogenase, is reported. This acyl enzyme undergoes all of the catalyzed reactions characteristic of the intermediate of the physiological acyl enzyme, 3-phospho-D-glyceroyl-glyceraldehyde-3-phosphate dehydrogenease. The rates of reactions of both these acyl enzymes depend strongly on the extent of interaction of the acyl enzyme with the oxidized coenzyme, NAD(+), even where the "redox" properties of the coenzyme are not required. Likewise, the spectral properties of chromophoric acyl enzyme are affected by the extent of bound NAD. Under the pseudophysiological conditions reported herein, there is a stoichiometric limitation of two furylacryloyl-acyl groups per enzyme molecule containing four covalently-equivalent subunits. The binding of NAD both to the apoenzyme and to the diacyl enzyme is heterogeneous: at low extents of NAD occupancy, NAD binding is stronger. The binding to acyl enzyme can be quantitatively described by an enzyme model involving a tetramer with 2-fold symmetry, and consequently containing equal numbers of two classes of sites. NAD binding to difurylacryloyl-enzyme occurs virtually discretely, first to the two unmodified (tight-binding) sites, followed by looser binding to the two acyl-sites. NAD occupancy at these latter sites transforms the chromophoric acyl spectrum from that characteristic of a model furylacryloyl-thiol ester in H(2)O to a highly perturbed furylacryloyl spectrum characteristic of monomeric native "active-thiol" furylacryloyl-enzymes. Likewise the acyl reactivity towards arsenolysis depends on the extent of NAD bound to the loose sites. Elimination of the tight binding of NAD to the difurylacryloyl enzyme tetramer by alkylation of the remaining two free SH groups with iodoacetate has no apparent influence on the NAD-dependent furylacryloyl-spectral perturbation at the "two equivalent acyl sites," even though it eliminates the apparent "negative cooperativity" in NAD binding.
报道了一种活性位点特异性发色酰基酶——鲟鱼β-(2-呋喃基)-丙烯酰甘油醛-3-磷酸脱氢酶的吸收光谱。这种酰基酶经历了生理酰基酶中间体——3-磷酸-D-甘油酰甘油醛-3-磷酸脱氢酶的所有催化反应特征。这两种酰基酶的反应速率都强烈依赖于酰基酶与氧化型辅酶NAD(+)的相互作用程度，即使在不需要辅酶“氧化还原”性质的情况下也是如此。同样，发色酰基酶的光谱性质也受结合NAD程度的影响。在本文报道的假生理条件下，每个含有四个共价等效亚基的酶分子存在两个呋喃丙烯酰酰基的化学计量限制。NAD与脱辅酶和二酰基酶的结合都是异质的：在NAD占据程度较低时，NAD结合更强。与酰基酶的结合可以通过一个涉及具有2重对称性的四聚体的酶模型进行定量描述，因此该模型包含数量相等的两类位点。NAD与二呋喃丙烯酰酶的结合实际上是离散发生的，首先结合到两个未修饰的（紧密结合）位点，随后较松散地结合到两个酰基位点。NAD在这些后一位点的占据将发色酰基光谱从H₂O中模型呋喃丙烯酰硫酯的特征光谱转变为单体天然“活性硫醇”呋喃丙烯酰酶特有的高度扰动的呋喃丙烯酰光谱。同样，酰基对砷解的反应性取决于结合到松散位点的NAD程度。用碘乙酸烷基化剩余的两个游离SH基团，消除NAD与二呋喃丙烯酰酶四聚体的紧密结合，对“两个等效酰基位点”处NAD依赖性呋喃丙烯酰光谱扰动没有明显影响，尽管它消除了NAD结合中明显的“负协同性”。
