Characterization of the S3 subsite specificity of cathepsin B.
Five synthetic substrates containing different amino acid residues at the P3 position (acetyl-X-Arg-Arg-AMC, where X is Gly, Glu, Arg, Val, and Tyr and where AMC represents 7-amindo-4-methylcoumarin) were used to investigate the S3 subsite specificity of cathepsin B. At pH 6.0, the specificity constant, kcat/Km, for tripeptide substrate hydrolysis was observed to increase in the order Glu < Gly < Arg < Val < Tyr. Molecular modeling studies of substrates containing a P3 Glu, Arg, or Tyr covalently bound as the tetrahedral intermediate to the enzyme suggest that the specificity for a P3 Tyr is because of a favorable aromatic-aromatic interaction with Tyr75 on the enzyme as well as a possible H bond between the P3 Tyr hydroxyl and the side chain carboxyl of Asp69.
Taralp
A
A
Department of Chemistry, University of Ottawa, Ontario, Canada.
Kaplan
H
H
Sytwu
I I
II
Vlattas
I
I
Bohacek
R
R
Knap
A K
AK
Hirama
T
T
Huber
C P
CP
Hasnain
S
S
D000595:Amino Acid Sequence; D000818:Animals; D002401:Cathepsin B / Q000737:chemistry / Q000378:metabolism*; D003374:Coumarins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D051381:Rats; D013379:Substrate Specificity; D013816:Thermodynamics
D003374:Coumarins; D009842:Oligopeptides; C094907:acetyl-arginyl-arginyl-arginyl-amidomethylcoumarin; C094906:acetyl-valyl-arginyl-arginyl-amidomethylcoumarin; D002401:Cathepsin B