Structural investigations of the active-site mutant Asn156Ala of outer membrane phospholipase A: Function of the Asn-His interaction in the catalytictriad

Outer membrane phospholipase A (OMPLA) from Escherichia coli is an integral -membrane enzyme with a unique His-Ser-Asn catalytic triad. In serine prote ases and serine esterases usually an Asp occurs in the catalytic triad; its role has been the subject of much debate. Here the role of the uncharged a sparagine in the active site of OMPLA is investigated by structural charact erization of the Asnl56Ala mutant. Asparagine 156 is not involved in mainta ining the overall active-site configuration and does not contribute signifi cantly to the thermal stability of OMPLA. The active-site histidine retains an active conformation in the mutant notwithstanding the loss of the hydro gen bond to the asparagine side chain. Instead, stabilization of the correc t tautomeric form of the histidine can account for the observed decrease in activity of the Asnl56Ala mutant.