Essential Lysine Residues in the N-Terminal and the C-Terminal Domain of Human Adenylate Kinase Interact with Adenine Nucleotides As Found by Site-Directed Random Mutagenesis 详细信息    查看全文

• 作者：Takanori Ayabe ; Hitoshi Takenaka ; Osamu Takenaka ; Michihiro Sumida ; Hideharu Maruyama ; Toshio Onitsuka ; Koichiro Shibata ; Seiichi Uesugi ; and Minoru Hamada
• 刊名：Biochemistry
• 出版年：1997
• 出版时间：April 1, 1997
• 年：1997
• 卷：36
• 期：13
• 页码：4027 - 4033
• 全文大小：147K
• 年卷期：v.36,no.13(April 1, 1997)
• ISSN：1520-4995

文摘

To elucidate the minimum requirement of amino acid residues forthe active center in humanadenylate kinase (hAK1), we carried out random site-directedmutagenesis of key lysine residues (K9,K21, K27, K31, K63, K131, and K194), which were conserved in mammalianAK1 species, with thepMEX8-hAK1 plasmid [Ayabe, T., et al. (1996) Biochem. Mol. Biol.Int. 38, 373-381]. Twentydifferentmutants were obtained and analyzed by steady-state kinetics, and allmutants showed activity loss by K_mand/or k_cat effects onMgATP^2-, AMP^2-, orboth. The results have led to the following conclusions.(1)Lys9 would appear to interact with bothMgATP^2- and AMP^2-but to a larger extent than withAMP^2-.(2) Lys21 is likely to play a role in substrate binding of bothMgATP^2- and AMP^2-but more stronglyaffects MgATP^2-. (3) Lys27 and Lys131would appear to play a functional role in catalysis byinteractingstrongly with MgATP^2-. (4) Lys31 wouldappear to interact with MgATP^2- andAMP^2- at theMgATP^2-site. (5) Lys63 would be more likely to interact withMgATP^2- than withAMP^2-. (6) Lys194 in theflanking C-terminal domain would appear to interact not only withMgATP^2- but also withAMP^2- at theMgATP^2- site by stabilizing substratebinding. The loss of the positively charged -amino groupoflysine affects both the affinity for the substrate and the catalyticefficiency. Hence, hydrophilic lysineresidues in hAK1 would appear to be essential for substrate-enzymeinteraction with the coordination ofsome arginine residues, reported previously [Kim, H. J., et al. (1990)Biochemistry 29, 1107-1111].