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 -