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 Humicol a lanuginosa lipase (HII) and the inactive mutant S146A (active Ser146 substituted with Ala) using fluorescence spectroscopy and molecular d ynamics simulations, respectively. Hll and S146A show significantly di fferent binding behavior for phosphatidylcholine (PC) and phosphatidyl glycerol (PG) liposomes. Generally, higher binding affinity is observe d for Hll than the S146A mutant. Furthermore,depending on the matrix, the addition of the transition state analogue benzene boronic acid inc reases the binding affinity of S146A, whereas only small changes are o bserved for I-Ill suggesting that the active site Lid in the latter op ens more easily and hence more lipase molecules are bound to the lipos omes. These observations are in agreement with molecular dynamics simu lations 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 S 146A than in Hll. Th ese findings reveal that the single mutation (S146A) of the active sit e serine leads to substantial conformational alterations in the H. lan uginosa Lipase and different binding affinities.