Active serine involved in the stabilization of the active site loop in the Humicola lanuginosa lipase.
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.
Peters
G H
GH
Chemistry Department III, H.C. Orsted Institutet, University of Copenhagen, Denmark. ghp@st.ki.ku.dk
Svendsen
A
A
Langberg
H
H
Vind
J
J
Patkar
S A
SA
Toxvaerd
S
S
Kinnunen
P K
PK
D000409:Alanine / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D004134:Dimyristoylphosphatidylcholine / Q000737:chemistry / Q000378:metabolism; D004795:Enzyme Stability; D008049:Lipase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D003904:Mitosporic Fungi / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010715:Phosphatidylglycerols / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary; D012694:Serine / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D008081:Liposomes; D010715:Phosphatidylglycerols; D012694:Serine; C002773:dimyristoylphosphatidylglycerol; D008049:Lipase; D000409:Alanine; D004134:Dimyristoylphosphatidylcholine