Active serine involved in the stabilization of the active site loop in the Humicola lanuginosa lipase
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Active serine involved in the stabilization of the active site loop in the Humicola lanuginosa lipase. / Peters, G H; Svendsen, A; Langberg, Henning; Vind, J; Patkar, S A; Toxvaerd, S; Kinnunen, P K.
In: Biochemistry, Vol. 37, No. 37, 1998, p. 12375-83.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Active serine involved in the stabilization of the active site loop in the Humicola lanuginosa lipase
AU - Peters, G H
AU - Svendsen, A
AU - Langberg, Henning
AU - Vind, J
AU - Patkar, S A
AU - Toxvaerd, S
AU - Kinnunen, P K
PY - 1998
Y1 - 1998
N2 - We have investigated the binding properties of and dynamics in Humicola lanuginosa lipase (Hll) and the inactive mutant S146A (active Ser146 substituted with Ala) using fluorescence spectroscopy and molecular dynamics simulations, respectively. Hll and S146A show significantly different binding behavior for phosphatidylcholine (PC) and phosphatidylglycerol (PG) liposomes. Generally, higher binding affinity is observed for Hll than the S146A mutant. Furthermore, depending on the matrix, the addition of the transition state analogue benzene boronic acid increases the binding affinity of S146A, whereas only small changes are observed for Hll suggesting that the active site lid in the latter opens more easily and hence more lipase molecules are bound to the liposomes. These observations are in agreement with molecular dynamics simulations and subsequent essential dynamics analyses. The results reveal that the hinges of the active site lid are more flexible in the wild-type Hll than in S146A. In contrast, larger fluctuations are observed in the middle region of the active site loop in S146A than in Hll. These findings reveal that the single mutation (S146A) of the active site serine leads to substantial conformational alterations in the H. lanuginosa lipase and different binding affinities.
AB - We have investigated the binding properties of and dynamics in Humicola lanuginosa lipase (Hll) and the inactive mutant S146A (active Ser146 substituted with Ala) using fluorescence spectroscopy and molecular dynamics simulations, respectively. Hll and S146A show significantly different binding behavior for phosphatidylcholine (PC) and phosphatidylglycerol (PG) liposomes. Generally, higher binding affinity is observed for Hll than the S146A mutant. Furthermore, depending on the matrix, the addition of the transition state analogue benzene boronic acid increases the binding affinity of S146A, whereas only small changes are observed for Hll suggesting that the active site lid in the latter opens more easily and hence more lipase molecules are bound to the liposomes. These observations are in agreement with molecular dynamics simulations and subsequent essential dynamics analyses. The results reveal that the hinges of the active site lid are more flexible in the wild-type Hll than in S146A. In contrast, larger fluctuations are observed in the middle region of the active site loop in S146A than in Hll. These findings reveal that the single mutation (S146A) of the active site serine leads to substantial conformational alterations in the H. lanuginosa lipase and different binding affinities.
KW - Alanine
KW - Binding Sites
KW - Dimyristoylphosphatidylcholine
KW - Enzyme Stability
KW - Lipase
KW - Liposomes
KW - Mitosporic Fungi
KW - Models, Molecular
KW - Mutagenesis, Site-Directed
KW - Phosphatidylglycerols
KW - Protein Structure, Secondary
KW - Serine
KW - Spectrometry, Fluorescence
KW - Thermodynamics
U2 - 10.1021/bi972883l
DO - 10.1021/bi972883l
M3 - Journal article
C2 - 9730809
VL - 37
SP - 12375
EP - 12383
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 37
ER -
ID: 155219