Cysteine 904 is required for maximal insulin degrading enzyme activity and polyanion activation.
Cysteine residues in insulin degrading enzyme have been reported as non-critical for its activity. We found that converting the twelve cysteine residues in rat insulin degrading enzyme (IDE) to serines resulted in a cysteine-free form of the enzyme with reduced activity and decreased activation by polyanions. Mutation of each cysteine residue individually revealed cysteine 904 as the key residue required for maximal activity and polyanion activation, although other cysteines affect polyanion binding to a lesser extent. Based on the structure of IDE, Asn 575 was identified as a potential hydrogen bond partner for Cys904 and mutation of this residue also reduced activity and decreased polyanion activation. The oligomerization state of IDE did not correlate with its activity, with the dimer being the predominant form in all the samples examined. These data suggest that there are several conformational states of the dimer that affect activity and polyanion activation.
Department of Molecular and Cellular Biochemistry and the Center for Structural Biology, University of Kentucky, Lexington, Kentucky, USA.
Eun Suk
ES
Song
Manana
M
Melikishvili
Michael G
MG
Fried
Maria A
MA
Juliano
Luiz
L
Juliano
David W
DW
Rodgers
Louis B
LB
Hersh
D000818:Animals; D002460:Cell Line; D003545:Cysteine / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004789:Enzyme Activation; D007339:Insulysin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D017354:Point Mutation; D000071228:Polyelectrolytes; D011108:Polymers / Q000378:metabolism*; D011487:Protein Conformation; D055503:Protein Multimerization; D051381:Rats; D013379:Substrate Specificity
D003545:Cysteine; D007339:Insulysin; D000071228:Polyelectrolytes; D011108:Polymers