Kurokawa Y, Watanabe A, Yoshimura T, Esaki N, Soda K
Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto, 611-0011, Japan.
J Biochem. 1998 Dec 1;124(6):1163-9. doi: 10.1093/oxfordjournals.jbchem.a022234.
The pyridoxal form of alanine racemase of Bacillus stearothermophilus was converted to the pyridoxamine form by incubation with its natural substrate, D- or L-alanine, under acidic conditions: the enzyme loses its racemase activity concomitantly. The pyridoxamine form of the enzyme returned to the pyridoxal form by incubation with pyruvate at alkaline pH. Thus, alanine racemase catalyzes transamination as a side function. In fact, the apo-form of the enzyme abstracted tritium from [4'-3H]pyridoxamine in the presence of pyruvate. A mutant enzyme containing alanine substituted for Lys39, whose epsilon-amino group forms a Schiff base with the C4' aldehyde of pyridoxal 5'-phosphate in the wild-type enzyme, was inactive as a catalyst for racemization as well as transamination. However, when methylamine was added to the mutant enzyme, it became active in both reactions. These results suggest that the epsilon-amino group of Lys39 participates in both racemization and transamination when catalyzed by the wild-type enzyme.
嗜热脂肪芽孢杆菌丙氨酸消旋酶的吡哆醛形式在酸性条件下与天然底物D-或L-丙氨酸一起温育时会转化为吡哆胺形式：与此同时，该酶失去其消旋酶活性。通过在碱性pH下与丙酮酸一起温育，该酶的吡哆胺形式又恢复为吡哆醛形式。因此，丙氨酸消旋酶作为一种次要功能催化转氨作用。事实上，在丙酮酸存在的情况下，该酶的脱辅基形式从[4'-3H]吡哆胺中提取了氚。一种含有取代赖氨酸39的丙氨酸的突变酶，其ε-氨基在野生型酶中与5'-磷酸吡哆醛的C4'醛形成席夫碱，作为消旋化和转氨作用的催化剂是无活性的。然而，当向突变酶中加入甲胺时，它在这两个反应中都变得有活性。这些结果表明，野生型酶催化时，赖氨酸39的ε-氨基参与消旋化和转氨作用。
