Identification of the role of a cysteine-rich region of PC6B by determining the enzymatic characteristics of its mutants.
In the proprotein convertases family, mouse PC6B (mPC6B) has a very large cysteine-rich region (CRR), consisting of 22 tandem cysteine-rich (Cys-rich) repeated segments. The role of this region remains elusive. In this report, to get insight on the possible role of the CRR, we constructed four truncated mPC6B mutant genes with 0, 5, 11, and 22 Cys-rich repeated segments remaining; using the baculovirus-expression system and a simple purification method, we obtained four enzyme mutants of mPC6B. By determining their optimal pH and calcium ion concentration for enzymatic activity and their thermal stability, we found that CRR did not affect pH optimum and Ca2+ optimum compared with the p-domain. However, CRR acted as a stabilizing domain in addition to the p-domain. By kinetic analyses of four mutants, we found that the long Cys-rich repeats in the native form of mPC6B reduced its Vmax. These facts suggest that CRR acts as an important part of functional domain.
Wang
Lie
L
Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yueyang Road, Shanghai, 200031, P.R. China.
Yang
Guanzhen
G
Wu
Xiangfu
X
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D003545:Cysteine; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D051379:Mice; D009154:Mutation; D045702:Proprotein Convertase 5 / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary
D045702:Proprotein Convertase 5; C473805:propeptide convertase 5, mouse; D003545:Cysteine; D002118:Calcium