Identification of reactive site of a proteinaceous metalloproteinase inhibitor from Streptomyces nigrescens TK-23.
Streptomyces metalloproteinase inhibitor (SMPI), isolated from Streptomyces nigrescens TK-23, is a small proteinaceous metalloproteinase inhibitor consisting of 102 amino acid residues and two disulfide bridges. SMPI specifically inhibits metalloproteinases such as thermolysin. After prolonged incubation with a catalytic amount of thermolysin, it is cleaved at Cys64-Val65 [Murai, H., Hara, S., Ikenaka, T., Oda, K., and Murao, S. (1985) J. Biochem. 97, 173-180]. Hence, for identification of the reactive site, mutants were constructed by substituting Val65 with various amino acid residues (Leu, Ile, Phe, Tyr, Gly, Ser, Lys, and Glu). The mutants were analyzed for inhibitory activity. Among them, V65I, V65L, V65F, and V65Y retained strong inhibitory activity, whereas V65S, V65G, V65K, and V65E showed very weak inhibitory activity against thermolysin. The Ki values were found to be of the order of 10(10) M by using a fluorogenic substrate, MOCAc-Pro-Leu-Gly-Leu-A2pr(Dnp)-Ala-Arg-NH2. In addition, susceptibility to enzyme degradation was analyzed by means of limited proteolysis with thermolysin. Mutants which retained strong inhibitory activity were cleaved by thermolysin only at the reactive site, in the same way as native SMPI. The mutants which showed weak inhibitory activity underwent rapid degradation. These results were consistent with the substrate specificity of thermolysin. Based on these results, the reactive site of SMPI was identified as Cys64-Val65.
Seeram
S S
SS
Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology.
Hiraga
K
K
Saji
A
A
Tashiro
M
M
Oda
K
K
D001426:Bacterial Proteins / Q000737:chemistry*; D001665:Binding Sites; D002942:Circular Dichroism; D006868:Hydrolysis; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors*; D016296:Mutagenesis; D011480:Protease Inhibitors / Q000737:chemistry*; D013302:Streptomyces / Q000737:chemistry*; D013820:Thermolysin / Q000037:antagonists & inhibitors
D001426:Bacterial Proteins; D011480:Protease Inhibitors; C056999:metalloproteinase inhibitor, Streptomyces; D008666:Metalloendopeptidases; D013820:Thermolysin