Avaeva S M, Rodina E V, Kurilova S A, Nazarova T I, Vorobyeva N N
A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Russian Federation.
FEBS Lett. 1996 Aug 26;392(2):91-4. doi: 10.1016/0014-5793(96)00791-0.
Asp-42 located in the active site of E. coli inorganic pyrophosphatase (PPase) has been substituted by Asn by site-directed mutagenesis. This resulted in a 3-fold increase in hydrolytic activity measured under optimal conditions, a 15.5-fold increase in the Km value and retention of the pK values of groups for enzyme and enzyme-substrate complex. The active site of the enzyme contains 4 metal binding centers (I-IV) [Harutyunyan et al. (1996) Eur. J. Biochem., in press]. Asp-42 is located near centers II and IV. The D42N replacement had no effect on Mg2+ binding with center II. At the same time, occupation of center IV eliminates the inhibition of inorganic pyrophosphate hydrolysis by high Mg2+ concentrations typical of wild-type PPase. It is proposed that the increase in activity and decrease in affinity for substrate of the D42N PPase results from changes in Mg2+ binding to center IV. The Mg2+ binding centers of E. coli PPase are lined up in filling order.
通过定点诱变，大肠杆菌无机焦磷酸酶（PPase）活性位点中的天冬氨酸-42（Asp-42）已被天冬酰胺（Asn）取代。这导致在最佳条件下测得的水解活性提高了3倍，米氏常数（Km）值提高了15.5倍，并且酶及酶-底物复合物基团的pK值保持不变。该酶的活性位点包含4个金属结合中心（I-IV）[哈鲁秋尼扬等人（1996年），《欧洲生物化学杂志》，即将发表]。Asp-42位于中心II和IV附近。D42N替换对Mg2+与中心II的结合没有影响。与此同时，中心IV被占据消除了野生型PPase典型的高Mg2+浓度对无机焦磷酸水解的抑制作用。有人提出，D42N PPase活性的增加和对底物亲和力的降低是由于Mg2+与中心IV结合的变化所致。大肠杆菌PPase的Mg2+结合中心按填充顺序排列。
