PHOSPHOLIPASE A(2) ENGINEERING - THE ROLES OF DISULFIDE BONDS IN STRUCTURE, CONFORMATIONAL STABILITY, AND CATALYTIC FUNCTION

Site-directed mutagenesis was used to probe the contribution of each of the seven disulfide bonds of bovine pancreatic phospholipase A(2) (PLA2, overexpressed in Escherichia coli) to the structure, conformational stability, and catalytic function of the enzyme. Each of the seven disulfide bonds, C11-C77, C27-C123, C29-C45, C44-C105, C51-C98, C61-C91, and C84-C96, was deleted separately by changing both cysteine (C) residues to alanine (A). The structural properties of the mutants were analyzed by 1D and 2D proton NMR, the conformational stability by guanidine hydrochloride-induced denaturation, and the catalytic property by measuring kinetic parameters toward DC8PC (1,2-dioctanoyl-sn-glycero-3-phosphocholine) micelles. The results led to the following significant findings: (i) All but one (C84A-C96A) mutants have been refolded and purified by use of the same procedure for wild-type PLA2. Thus, the disulfide bonds are generally not important to the folding pathway of PLA2. (ii) The disulfide bond C11-C77 is most important to the conformation and conformational stability of the enzyme since deletion of this disulfide bond resulted in greatly perturbed NMR properties and in a decrease of 6.2 kcal/mol in conformational stability. However, the C11A-C77A mutant displayed little change in catalytic function. (iii) The effects of deleting disulfide bonds on the catalytic function of PLA2 are small, except the disulfide bond C29-C45 which connects the calcium binding loop with the helix C. However, the conformation and conformational stability of the C29A-C45A mutant are essentially unperturbed. (iv) The calcium binding affinity of the C29A-C45A mutant was found to decrease by a factor of 10 or greater. This could be the cause for the perturbation in kinetic behavior. (v) Deletion of the C27-C123 disulfide bond caused an unexpected increase in the conformational stability of the enzyme by 2.4 kcal/mol. The overall results are discussed in relation to the structure-function relationship of PLA2 and the roles of disulfide bonds in protein structures.