Mammalian spermidine synthase--identification of cysteine residues and investigation of the putrescine binding site--.
Homology modeling and inhibitory studies using substrate analogs were undertaken to construct a possible three-dimensional structure, including the putrescine-binding site, of rat spermidine synthase based on its primary sequence. Of the ten cysteine residues of the enzyme, six residues were chemically determined as sulfhydryl; similarly, one residue (C25) was determined as the disulfide. Using the model obtained from the Swiss-Model protein-modeling server, and based on the crystal structure of the Thermotoga maritima enzyme, the three remaining residues were assigned as sulfhydryl. Discussions are presented on the counterpart of the C25 residue, based on the apparent role of the bacterial N-terminal peptide region in reinforcing the binding between protomers in a functional oligomeric form. The active sites of the bacterial and mammalian versions of the enzyme were very similar. The putrescine-binding site of the rat enzyme was investigated using IC(50) values of the analogs of two known potent inhibitors, n-butylamine and trans-4-methylcyclohexylamine (4MCHA). Our results indicated that 5-amino-1-pentene and 4MCHA possess comparable inhibitory activities towards the enzyme.
Goda
Hitomi
H
Faculty of Pharmaceutical Sciences, Josai University, 1-1 Keyakidai, Sakado, Saitama 350-0295, Japan.
Watanabe
Toshiko
T
Takeda
Noboru
N
Kobayashi
Masaki
M
Wada
Makiko
M
Hosoda
Harumi
H
Shirahata
Akira
A
Samejima
Keijiro
K
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D002082:Butylamines / Q000737:chemistry / Q000494:pharmacology; D003514:Cyclohexylamines / Q000737:chemistry / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry*; D008297:Male; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011700:Putrescine / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011436:Spermidine Synthase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism
D002082:Butylamines; D003514:Cyclohexylamines; C059116:4-methylcyclohexylamine; D011436:Spermidine Synthase; D003545:Cysteine; C032462:n-butylamine; D011700:Putrescine