Sakai K, Hasumi K, Endo A
Department of Agricultural and Biological Chemistry, Tokyo Noko University, Japan.
Biochim Biophys Acta. 1988 Feb 10;952(3):297-303. doi: 10.1016/0167-4838(88)90130-6.
Koningic acid, a sesquiterpene antibiotic, is a specific inhibitor of the enzyme glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12). In the presence of 3 mM of NAD+, koningic acid irreversibly inactivated the enzyme in a time-dependent manner. The pseudo-first-order rate constant for inactivation (kapp) was dependent on koningic acid concentration in saturate manner, indicating koningic acid and enzyme formed a reversible complex prior to the formation of an inactive, irreversible complex; the inactivation rate (k 3) was 5.5.10(-2) s-1, with a dissociation constant for inactivation (Kinact) of 1.6 microM. The inhibition was competitive against glyceraldehyde 3-phosphate with a Ki of 1.1 microM, where the Km for glyceraldehyde 3-phosphate was 90 microM. Koningic acid inhibition was uncompetitive with respect to NAD+. The presence of NAD+ accelerated the inactivation. In its absence, the charcoal-treated NAD+-free enzyme showed a 220-fold decrease in apparent rate constant for inactivation, indicating that koningic acid sequentially binds to the enzyme next to NAD+. The enzyme, a tetramer, was inactivated when maximum two sulfhydryl groups, possibly cysteine residues at the active sites of the enzyme, were modified by the binding of koningic acid. These observations demonstrate that koningic acid is an active-site-directed inhibitor which reacts predominantly with the NAD+-enzyme complex.
koningic酸是一种倍半萜抗生素，是甘油醛-3-磷酸脱氢酶（D-甘油醛-3-磷酸：NAD+氧化还原酶（磷酸化），EC 1.2.1.12）的特异性抑制剂。在存在3 mM NAD+的情况下，koningic酸以时间依赖性方式不可逆地使该酶失活。失活的伪一级速率常数（kapp）以饱和方式依赖于koningic酸浓度，表明koningic酸与酶在形成无活性的不可逆复合物之前形成了可逆复合物；失活速率（k3）为5.5×10-2 s-1，失活解离常数（Kinact）为1.6 μM。该抑制作用对甘油醛-3-磷酸具有竞争性，Ki为1.1 μM，其中甘油醛-3-磷酸的Km为90 μM。koningic酸抑制作用对NAD+而言是非竞争性的。NAD+的存在加速了失活。在其不存在的情况下，经活性炭处理的无NAD+酶的失活表观速率常数降低了220倍，表明koningic酸依次在NAD+旁边与酶结合。该酶是一种四聚体，当最多两个巯基（可能是酶活性位点的半胱氨酸残基）通过koningic酸的结合被修饰时，酶被失活。这些观察结果表明，koningic酸是一种活性位点导向的抑制剂，主要与NAD+-酶复合物反应。
