人腺苷酸激酶突变酶的定点诱变及稳态动力学分析

Site-directed mutagenesis and steady-state kinetic analysis of mutant enzymes of human adenylate kinase.

作者信息

Ayabe T, Park S K, Nagahama H, Maruyama H, Sumida M, Takenaka H, Takenaka O, Onitsuka T, Hamada M

机构信息

Department of Hygiene, Miyazaki Medical College, Japan.

出版信息

Biochem Mol Biol Int. 1998 Nov;46(4):673-80. doi: 10.1080/15216549800204202.

DOI:10.1080/15216549800204202

PMID:9844727

Abstract

Site-directed mutagenesis of human adenylate kinase (AK) was carried out on residues His36, Lys55, and the C-terminal segment (Val182, V186, and Leu193). Five mutants [(H36T, K55G, V182G, V186S, and L193Stop (deletion of residues 193-194)] were generated and analyzed by steady-state kinetics. H36T, K55G, and L193Stop mutants showed an increase of K(m) values (19.8-, 19.7-, and 11.3-fold) for AMP2- compared to that for the wild-type enzyme, and these residues appeared to interact with AMP2-. V182G showed an increased K(m) value (7.4-fold) for MgATP2-. Therefore, V182 may be essential for interaction with MgATP2-. V186S increased the K(m) value (7.0- and 7.5-fold) for MgATP2- and AMP2-. V186 may thus interact with both substrates. The C-terminal domain of AK appears to be essential for MgATP2- and AMP2- binding.

摘要

对人腺苷酸激酶（AK）的组氨酸36、赖氨酸55和C末端片段（缬氨酸182、缬氨酸186和亮氨酸193）进行了定点诱变。生成了五个突变体[（H36T、K55G、V182G、V186S和L193Stop（缺失193 - 194位残基）]，并通过稳态动力学进行分析。与野生型酶相比，H36T、K55G和L193Stop突变体对AMP2 - 的K(m)值增加（分别为19.8倍、19.7倍和11.3倍），这些残基似乎与AMP2 - 相互作用。V182G对MgATP2 - 的K(m)值增加（7.4倍）。因此，缬氨酸182可能对与MgATP2 - 的相互作用至关重要。V186S对MgATP2 - 和AMP2 - 的K(m)值增加（分别为7.0倍和7.5倍）。因此，缬氨酸186可能与两种底物都相互作用。AK的C末端结构域似乎对MgATP2 - 和AMP2 - 的结合至关重要。