Two inhibitor molecules bound in the active site of Pseudomonas sedolisin: a model for the bi-product complex following cleavage of a peptide substrate.
High-resolution crystallographic analysis of a complex of the serine-carboxyl proteinase sedolisin with pseudo-iodotyrostatin revealed two molecules of this inhibitor bound in the active site of the enzyme, marking subsites from S3 to S3('). The mode of binding represents two products of the proteolytic reaction. Substrate specificity of sedolisin was investigated using peptide libraries and a new peptide substrate for sedolisin, MCA-Lys-Pro-Pro-Leu-Glu#Tyr-Arg-Leu-Gly-Lys(DNP)-Gly, was synthesized based on the results of the enzymatic and crystallographic studies and was shown to be efficiently cleaved by the enzyme. The kinetic parameters for the substrate, measured by the increase in fluorescence upon relief of quenching, were: k(cat)=73+/-5 s(-1), K(m)=0.12+/-0.011 microM, and k(cat)/K(m)=608+/-85 s(-1)microM(-1).
Wlodawer
Alexander
A
Protein Structure Section, Macromolecular Crystallography Laboratory, National Cancer Institute at Frederick, Frederick, MD 21702, USA. wlodawer@ncifcrf.gov
Li
Mi
M
Gustchina
Alla
A
Oyama
Hiroshi
H
Oda
Kohei
K
Beyer
Bret B
BB
Clemente
Jose
J
Dunn
Ben M
BM
D001665:Binding Sites; D002268:Carboxypeptidases / Q000737:chemistry*; D018360:Crystallography, X-Ray; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D011549:Pseudomonas / Q000378:metabolism*; D013379:Substrate Specificity
D019151:Peptide Library; D010455:Peptides; D002268:Carboxypeptidases; C479198:sedolisin