The importance of Loop 7 for the activity of calcineurin.
Calcineurin (CN) is a heterodimer composed of a catalytic subunit (CNA) and a regulatory subunit (CNB). Loop 7 lies within the CNA catalytic domain. To investigate the role of Loop 7 in enzyme activity, we systematically examined all its residues by site-directed deletion mutation. Our results show that the Loop 7 residues are important for enzyme activity. Besides deleting residues V314, Y315 or N316, enzyme activity also increased dramatically when residues D313 or K318 were deleted. In contrast, almost all activity was lost when L312 or N317 were deleted. Ni2+ and Mn2+ were effective activators for all active mutants. However, whereas the wild-type enzyme was more efficiently activated by Ni2+ than by Mn2+ with 32P-labeled R(II) peptide as substrate, the reverse was true in all the mutants. We also found that the effect of Loop 7 on enzyme activity was substrate dependent, and involved interactions between Loop 7 residues and the unresolved part of the CN crystal structure near the auto-inhibitory domain and catalytic site.
Liu
Ping
P
Department of Biochemistry and Molecular Biology, Beijing Normal University, Beijing Key Laboratory, Beijing 100875, PR China.
Huang
Chao
C
Wang
Hai-long
HL
Zhou
Ke
K
Xiao
Fang-xiang
FX
Qun
Wei
W
D000818:Animals; D019703:Calcineurin / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020134:Catalytic Domain; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D051381:Rats; D017384:Sequence Deletion
D019703:Calcineurin