Mutational analysis of the reactive site loop of Streptomyces metalloproteinase inhibitor, SMPI.
Streptomyces metalloproteinase inhibitor (SMPI) is the only inhibitor to show "standard mechanism inhibition" against metalloproteinases. SMPI is a globular protein with an exposed loop containing the reactive site, C64-V65. To analyze the importance of basic residues in the reactive site loop of SMPI, mutants were constructed for R60, K61, and R66 (R60A, K61A, R66A, R60/K61A, 60/61/66A, and 60/61/66E). The mutants involving only R60, K61, and R60/K61 residues, respectively, showed strong inhibitory activity and were stable against enzyme activity. Both the triple mutants showed very weak inhibitory activity and underwent rapid degradation. The addition of basic residues to the loop (V62R and T63R) did not cause any further increase in inhibitory activity. These results suggest that basic residues in the reactive site loop play some role in maintaining a stable enzyme-inhibitor complex. The R66 mutant showed reduced activity and was rapidly degraded by enzymes. It was concluded that R66 is essential for maintaining a strong hydrophobic interaction with the S1' hydrophobic pocket of the enzyme. To investigate the roles of the disulfide bridge and the P68 residue near the reactive site, C64/69S and P68T mutants were constructed. These mutants showed very weak inhibitory activity and were rapidly degraded by enzymes. These results suggest that the disulfide bridge and P68 residue are very essential for SMPI to function as an inhibitor.
Hiraga
K
K
Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology, Kyoto, 606-8585, Japan.
Seeram
S S
SS
Tate
S
S
Tanaka
N
N
Kainosho
M
M
Oda
K
K
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D001665:Binding Sites; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors*; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D013302:Streptomyces / Q000737:chemistry*; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D004220:Disulfides; C056999:metalloproteinase inhibitor, Streptomyces; D008666:Metalloendopeptidases