Trommer W E, Huth H, Wenzel H R
Biochim Biophys Acta. 1979 Mar 16;567(1):49-59. doi: 10.1016/0005-2744(79)90171-2.
The formation of the ternary complex of lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) from pig heart and skeletal muscle with the adduct of pyruvate to NAD", spin-labeled at N6 was studied by ultraviolet spectroscopy and ESR techniques. According to ultraviolet measurements we found identical binding characteristics for the natural coenzyme and its spin-labeled analog. The rate by which the ESR signal of free spin-labeled NAD+ decreased upon addition of pyruvate to the binary complexes was substantially different in the two isozymes. With the heart type an initial drop followed by a further linear decrease, zero order in the enzyme and coenzyme concentration was observed. In case of the skeletal muscle isozyme no immediate reaction and a first order process occurred. The initial reaction can be attributed to a non-covalent enzyme/spin-labeled NAD+/pyruvate complex with a dissociation constant for pyruvate of 11 +/- 1 mM, thus explaining the well-known substrate inhibition in the heart isozyme above 2 mM pyruvate. The further reaction is then determined by the buffer dependent enolization of pyruvate. In the muscle isozyme formation of the covalent adduct is not assisted by prior binding of pyruvate in a non-covalent ternary complex and therefore the rate depends on the binary complex concentration.
利用紫外光谱法和电子自旋共振（ESR）技术，研究了猪心脏和骨骼肌中乳酸脱氢酶（L-乳酸：NAD⁺氧化还原酶，EC 1.1.1.27）与N⁶位自旋标记的丙酮酸与NAD⁺加合物形成的三元复合物。根据紫外测量结果，我们发现天然辅酶及其自旋标记类似物具有相同的结合特性。在两种同工酶中，向二元复合物中加入丙酮酸后，游离自旋标记的NAD⁺的ESR信号下降速率有很大差异。对于心脏型同工酶，观察到初始下降后进一步呈线性下降，在酶和辅酶浓度方面为零级反应。对于骨骼肌同工酶，未观察到立即反应，而是发生一级反应。初始反应可归因于一种非共价的酶/自旋标记的NAD⁺/丙酮酸复合物，丙酮酸的解离常数为11±1 mM，这就解释了心脏同工酶在丙酮酸浓度高于2 mM时众所周知的底物抑制现象。随后的反应则由缓冲液依赖的丙酮酸烯醇化作用决定。在肌肉同工酶中，丙酮酸在非共价三元复合物中的预先结合无助于共价加合物的形成，因此反应速率取决于二元复合物的浓度。
