Relationship between temporary inhibition and structure of disulfide-linkage analogs of marinostatin, a natural ester-linked protein protease inhibitor.
A 12-residue marinostatin [MST(1-12): (1)FATMRYPSDSDE(12)] which contains two ester linkages of Thr(3)-Asp(9) and Ser(8)-Asp(11) strongly inhibits subtilisin. In order to study the relationship between the inhibitory activity, structure, and stability of MST, MST analogs were prepared by changing ester linkages to a disulfide linkages. The analogs without the disulfide linkage between 3 and 9 positions lost their inhibitory activity. The K(i) value of 1SS(C(3)-C(9)) ((1)FACMRYPSCSDE(12)), which has a single disulfide linkage of Cys(3)-Cys(9) was comparable with those of MST(1-12) and MST-2SS ((1)FACMRYPCCSCE(12)), a doubly linked analog of Cys(3)-Cys(9) and Cys(8)-Cys(11). However, 1SS(C(3)-C(9)) and MST-2SS showed temporary inhibition, but not MST(1-12): These analogs were inactivated after incubation with subtilisin for 30 min, and were specifically hydrolyzed at the reactive site. (1)H NMR study showed that 1SS(C(3)-C(9)) has two conformations, which contain a cis- (70%) or trans- (30%) Pro residue, while MST-2SS as well as MST(1-12) takes a single conformation containing only a cis-Pro residue. Hydrogen-deuterium exchange rate of the Arg(5) (P1') NH proton of the MST analogs was about 100 times faster than that of MST(1-12). These results indicate that the linkage between the positions 8 and 11 plays a role for fixing the cis-conformation of the Pro(7) residue, and that the linkage between 3 and 9 is indispensable for the inhibition, but not enough for stable protease-inhibitor complex.
Department of Applied Biology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan.
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M
Taniguchi
K
K
Kamei
K
K
Kanaori
T
T
Koyama
T
T
Yasui
R
R
Takano
S
S
Harada
K
K
Tajima
C
C
Imada
S
S
Hara
D018528:ATP-Binding Cassette Transporters / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D041961:Deuterium Exchange Measurement; D004220:Disulfides / Q000737:chemistry; D004952:Esters / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular; D011392:Proline / Q000737:chemistry; D011480:Protease Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D011487:Protein Conformation; D011498:Protein Precursors / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D013329:Structure-Activity Relationship; D020860:Subtilisin / Q000037:antagonists & inhibitors*
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D004220:Disulfides; D004952:Esters; D011480:Protease Inhibitors; D011498:Protein Precursors; C117587:marinostatin L protein, Alteromonas; D011392:Proline; D020860:Subtilisin