TY - JOUR
T1 - Use of Site-Directed Mutagenesis To Identify Valine-573 in the S′1 Binding Site of Rat Neutral Endopeptidase 24.11 (Enkephalinase)
AU - Vijayaraghavan, Jayanthi
AU - Kim, Young Ae
AU - Jackson, Deborah
AU - Hersh, Louis B.
AU - Orlowski, Marian
PY - 1990/8/1
Y1 - 1990/8/1
N2 - On the basis of the identity of a segment of the amino acid sequence within the active site of the bacterial enzyme thermolysin and the mammalian enzyme neutral endopeptidase 24.11, the possible involvement of valine-573 of neutral endopeptidase 24.11 in substrate binding was investigated. Valine-573 was changed to leucine and to alanine by site-directed mutagenesis. The effect of these mutations on inhibitor binding and substrate catalysis was examined with a series of compounds containing variable P′1 residues. With a small P′1 residue such as alanine, both mutant enzymes exhibited kinetic properties essentially the same as the wild-type enzyme. However, with larger P′1 residues such as phenylalanine, tyrosine, and leucine, the Val573Leu mutant showed a 24–100-fold decrease in inhibitor affinity. Similarly substrates containing bulky P′1 residues showed a 10–40-fold decrease in Vmax with little change in Km. In contrast, the Val573Ala mutant showed only modest changes in terms of inhibitor binding or substrate turnover. These results support the proposed role of valine-573 as a part of the hydrophobic binding pocket, S′1 binding subsite, of neutral endopeptidase 24.11.
AB - On the basis of the identity of a segment of the amino acid sequence within the active site of the bacterial enzyme thermolysin and the mammalian enzyme neutral endopeptidase 24.11, the possible involvement of valine-573 of neutral endopeptidase 24.11 in substrate binding was investigated. Valine-573 was changed to leucine and to alanine by site-directed mutagenesis. The effect of these mutations on inhibitor binding and substrate catalysis was examined with a series of compounds containing variable P′1 residues. With a small P′1 residue such as alanine, both mutant enzymes exhibited kinetic properties essentially the same as the wild-type enzyme. However, with larger P′1 residues such as phenylalanine, tyrosine, and leucine, the Val573Leu mutant showed a 24–100-fold decrease in inhibitor affinity. Similarly substrates containing bulky P′1 residues showed a 10–40-fold decrease in Vmax with little change in Km. In contrast, the Val573Ala mutant showed only modest changes in terms of inhibitor binding or substrate turnover. These results support the proposed role of valine-573 as a part of the hydrophobic binding pocket, S′1 binding subsite, of neutral endopeptidase 24.11.
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U2 - 10.1021/bi00487a009
DO - 10.1021/bi00487a009
M3 - Article
C2 - 2261463
AN - SCOPUS:0024997577
SN - 0006-2960
VL - 29
SP - 8052
EP - 8056
JO - Biochemistry
JF - Biochemistry
IS - 35
ER -