Hydrogen bond residue positioning in the 599-611 loop of thimet oligopeptidase is required for substrate selection.
Thimet oligopeptidase (EC 3.4.24.15) is a zinc(II) endopeptidase implicated in the processing of numerous physiological peptides. Although its role in selecting and processing peptides is not fully understood, it is believed that flexible loop regions lining the substrate-binding site allow the enzyme to conform to substrates of varying structure. This study describes mutant forms of thimet oligopeptidase in which Gly or Tyr residues in the 599-611 loop region were replaced, individually and in combination, to elucidate the mechanism of substrate selection by this enzyme. Decreases in k(cat) observed on mutation of Tyr605 and Tyr612 demonstrate that these residues contribute to the efficient cleavage of most substrates. Modeling studies showing that a hinge-bend movement brings both Tyr612 and Tyr605 within hydrogen bond distance of the cleaved peptide bond supports this role. Thus, molecular modeling studies support a key role in transition state stabilization of this enzyme by Tyr605. Interestingly, kinetic parameters show that a bradykinin derivative is processed distinctly from the other substrates tested, suggesting that an alternative catalytic mechanism may be employed for this particular substrate. The data demonstrate that neither Tyr605 nor Tyr612 is necessary for the hydrolysis of this substrate. Relative to other substrates, the bradykinin derivative is also unaffected by Gly mutations in the loop. This distinction suggests that the role of Gly residues in the loop is to properly orientate these Tyr residues in order to accommodate varying substrate structures. This also opens up the possibility that certain substrates may be cleaved by an open form of the enzyme.
Bruce
Lisa A
LA
Chemistry Department, Wellesley College, MA 02481-8203, USA.
Sigman
Jeffrey A
JA
Randall
Danica
D
Rodriguez
Scott
S
Song
Michelle M
MM
Dai
Yi
Y
Elmore
Donald E
DE
Pabon
Amanda
A
Glucksman
Marc J
MJ
Wolfson
Adele J
AJ
D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D001920:Bradykinin / Q000378:metabolism; D002384:Catalysis; D006801:Humans; D006860:Hydrogen Bonding; D007700:Kinetics; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D013379:Substrate Specificity
D008666:Metalloendopeptidases; C022510:thimet oligopeptidase; D001920:Bradykinin