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 o f the seven disulfide bonds of bovine pancreatic phospholipase A(2) (P LA2, overexpressed in Escherichia coli) to the structure, conformation al stability, and catalytic function of the enzyme. Each of the seven disulfide bonds, C11-C77, C27-C123, C29-C45, C44-C105, C51-C98, C61-C9 1, and C84-C96, was deleted separately by changing both cysteine (C) r esidues to alanine (A). The structural properties of the mutants were analyzed by 1D and 2D proton NMR, the conformational stability by guan idine hydrochloride-induced denaturation, and the catalytic property b y measuring kinetic parameters toward DC8PC (1,2-dioctanoyl-sn-glycero -3-phosphocholine) micelles. The results led to the following signific ant findings: (i) All but one (C84A-C96A) mutants have been refolded a nd 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 confor mation and conformational stability of the enzyme since deletion of th is disulfide bond resulted in greatly perturbed NMR properties and in a decrease of 6.2 kcal/mol in conformational stability. However, the C 11A-C77A mutant displayed little change in catalytic function. (iii) T he effects of deleting disulfide bonds on the catalytic function of PL A2 are small, except the disulfide bond C29-C45 which connects the cal cium binding loop with the helix C. However, the conformation and conf ormational stability of the C29A-C45A mutant are essentially unperturb ed. (iv) The calcium binding affinity of the C29A-C45A mutant was foun d to decrease by a factor of 10 or greater. This could be the cause fo r the perturbation in kinetic behavior. (v) Deletion of the C27-C123 d isulfide bond caused an unexpected increase in the conformational stab ility of the enzyme by 2.4 kcal/mol. The overall results are discussed in relation to the structure-function relationship of PLA2 and the ro les of disulfide bonds in protein structures.