Cheng Z Q, McFadden B A
Department of Biochemistry and Biophysics, Washington State University, Pullman 99164-4660, USA.
Protein Eng. 1998 Jun;11(6):457-65. doi: 10.1093/protein/11.6.457.
The replacement of all 22 completely conserved glycine residues in the large subunit of ribulose bisphosphate carboxylase/oxygenase from Anacystis nidulans by directed mutagenesis is described. In each beta/alpha barrel of the large subunit there are 12 completely conserved glycines in six of eight loops at the C-termini of eight beta-strands and four in loops at N-terminal ends of the beta-strands. Two completely conserved glycines are also in each beta/alpha barrel backbone and four more are in a large N-terminal portion preceding the barrel in a given L subunit. Substitution of glycines in loops that are C-terminal to beta-strands by proline was more deleterious to carboxylase activity than that by alanine supporting the postulates that these loops contribute to catalysis and substrate binding and that in some cases the glycines may serve as hinges enabling movement of the loops. In contrast, substitution of glycines at the N-terminal ends of beta-strands in the beta/alpha barrel more often led to failure to detect L subunits or their assembly into L8S8 complex. Substitution of these and the other conserved glycines by proline was more deleterious to carboxylase activity than by alanine in enzymes that assembled.
本文描述了通过定向诱变替换集胞藻6803核酮糖二磷酸羧化酶/加氧酶大亚基中所有22个完全保守的甘氨酸残基的过程。在大亚基的每个β/α桶中，八个β链C末端的八个环中的六个环上有12个完全保守的甘氨酸，β链N末端的环上有四个。每个β/α桶主链中也有两个完全保守的甘氨酸，在给定L亚基中桶之前的大N末端部分还有四个。用脯氨酸取代β链C末端环中的甘氨酸对羧化酶活性的损害比用丙氨酸取代更大，这支持了这些环有助于催化和底物结合的假设，并且在某些情况下，甘氨酸可能充当使环移动的铰链。相比之下，在β/α桶中β链N末端的甘氨酸取代更常导致无法检测到L亚基或它们组装成L8S8复合物。在组装的酶中，用脯氨酸取代这些和其他保守的甘氨酸对羧化酶活性的损害比用丙氨酸更大。
