pH dependence studies provide insight into the structure and mechanism of thimet oligopeptidase (EC 3.4.24.15).
Thimet oligopeptidase (EC 3.4.24.15; TOP) is a Zn(II) endopeptidase implicated in physiological regulation of processes involving neuropeptides. The present study clarifies the active site structure and mechanism of catalysis of TOP. The enzyme exhibited a bell-shaped pH dependence of activity having an acidic limb due to a protonation event with a pK(a) of 5.7 and a basic limb with pK(a) of 8.8. The acidic limb can be attributed to protonation of a residue affecting k(cat) while the alkaline limb may be due to conformational change. Mutation of Tyr612 to Phe resulted in more than 400-fold decrease in activity. This result, supported by modeling studies, implicates Tyr612 in transition state stabilization analogous to the role of His231 of thermolysin.
Sigman
Jeffrey A
JA
Department of Chemistry, Wellesley College, Wellesley, MA 02481, USA.
Edwards
Sarah R
SR
Pabon
Amanda
A
Glucksman
Marc J
MJ
Wolfson
Adele J
AJ
D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000235:genetics / Q000502:physiology
D014443:Tyrosine; D008666:Metalloendopeptidases; C022510:thimet oligopeptidase