Chaidaroglou A, Brezinski D J, Middleton S A, Kantrowitz E R
Department of Chemistry, Boston College, Massachusetts 02167.
Biochemistry. 1988 Nov 1;27(22):8338-43. doi: 10.1021/bi00422a008.
The function of arginine residue 166 in the active site of Escherichia coli alkaline phosphatase was investigated by site-directed mutagenesis. Two mutant versions of alkaline phosphatase, with either serine or alanine in the place of arginine at position 166, were generated by using a specially constructed M13 phage carrying the wild-type phoA gene. The mutant enzymes with serine and alanine at position 166 have very similar kinetic properties. Under conditions of no external phosphate acceptor, the kcat for the mutant enzymes decreases by approximately 30-fold while the Km increases by less than 2-fold. When kinetic measurements are carried out in the presence of a phosphate acceptor, 1.0 M Tris, the kcat for the mutant enzymes is reduced by less than 3-fold, while the Km increases by more than 50-fold. For both mutant enzymes, in either the absence or the presence of a phosphate acceptor, the catalytic efficiency as measured by the kcat/Km ratio decreases by approximately 50-fold as compared to the wild type. Measurements of the Ki for inorganic phosphate show an increase of approximately 50-fold for both mutants. Phenylglyoxal, which inactivates the wild-type enzyme, does not inactivate the Arg-166----Ala enzyme. This result indicates that Arg-166 is the same arginine residue that when chemically modified causes loss of activity [Daemen, F.J.M., & Riordan, J.F. (1974) Biochemistry 13, 2865-2871]. The data reported here suggest that although Arg-166 is important for activity is not essential. The analysis of the kinetic data also suggests that the loss of arginine-166 at the active site of alkaline phosphatase has two different effects on the enzyme. First, the binding of the substrate, and phosphate as a competitive inhibitor, is reduced; second, the rate of hydrolysis of the covalent phosphoenzyme may be diminished.
通过定点诱变研究了大肠杆菌碱性磷酸酶活性位点中精氨酸残基166的功能。使用携带野生型phoA基因的特制M13噬菌体，产生了两个碱性磷酸酶突变体，在位置166处分别用丝氨酸或丙氨酸取代精氨酸。166位为丝氨酸和丙氨酸的突变酶具有非常相似的动力学性质。在没有外部磷酸受体的条件下，突变酶的kcat降低约30倍，而Km增加不到2倍。当在磷酸受体1.0 M Tris存在下进行动力学测量时，突变酶的kcat降低不到3倍，而Km增加超过50倍。对于这两种突变酶，无论有无磷酸受体，通过kcat/Km比值测量的催化效率与野生型相比降低约50倍。无机磷酸的Ki测量表明，两种突变体的Ki均增加约50倍。使野生型酶失活的苯乙二醛不会使Arg-166→Ala酶失活。该结果表明，Arg-166就是那个经化学修饰后会导致活性丧失的精氨酸残基[Daemen, F.J.M., & Riordan, J.F. (1974) Biochemistry 13, 2865 - 2871]。此处报道的数据表明，尽管Arg-166对活性很重要，但并非必不可少。动力学数据分析还表明，碱性磷酸酶活性位点处精氨酸-166的缺失对该酶有两种不同影响。首先，底物以及作为竞争性抑制剂的磷酸盐的结合减少；其次，共价磷酸酶的水解速率可能降低。
