The N-terminal residues of phospholipase A2 (PLA2) are believed to be involved in the hydrogen-bonding network, the interfacial binding site, or the hydrophobic channel. Site-directed mutants of bovine pancreatic PLA2 with substitutions at positions 2, 3, 4, 5, 6, and 9 were constructed to test the roles of these residues in the structure and function of PLA2. Nonconservative mutations of Phe-5 and Ile-9, which are located inside the hydrophobic channel, led to significant perturbations in the conformation and conformational stability. Kinetic studies also indicated that mutations at Ile-9 and Phe-5 caused significant decreases in the rate of hydrolysis toward micellar and vesicle substrates. Scooting mode kinetic analysis showed that the binding step of the mutant enzymes to the DC14PM (1,2-dimyristoyl-sn-glycero-3-phosphomethanol) vesicle interface is not significantly affected and that the perturbations in catalysis occur mainly in kcat at the interface. The results taken together suggest that the residues Ile-9 and Phe-5 are important for both structure and catalysis. The mutant W3A (Trp-3 to Ala) also showed decreased rates of hydrolysis but to a lesser extent than Ile-9 and Phe-5 mutants. In addition, the binding affinity of W3A to the surface of the vesicles (i.e., the E to E* step) has been perturbed to the extent that hopping between anionic vesicles has been observed. On the other hand, the mutants of Gln-4 and Asn-6, which are located at or near the surface, displayed structural and kinetic properties similar to those of the wild-type PLA2 with the exception of the highly hydrophilic lysine mutant. The X-ray structure of the Q4E mutant indicates that the overall structure, the catalytic triad, and the link between residue 4 and Asp-99 via hydrogen bonding through Ala-1 and the structural water remain the same as in the WT. Substitutions for Leu at position 2 showed an acyl chain length discrimination toward different substrates, which may reflect the contacting position(s) of the substrate acyl chain with Leu-2.

Site-directed mutagenesis was used to probe the contribution of each of the seven disulfide bonds of bovine pancreatic phospholipase A2 (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 was found to decrease by a factor of 10 or greater. (ABSTRACT TRUNCATED AT 250 WORDS)

To probe the role of the Asp-99. . .His-48 pair in phospholipase A, (PLA2) catalysis, the X-ray structure and kinetic characterization of the mutant Asp-99 + Asn-99 (D99N) of bovine pancreatic PLAZ was undertaken. Crystals of D99N belong to the trigonal space group P3121 and were isomorphous to the wild type (WT) (Noel J P et al., 1991, Biochemistry 30:11801-11811). The 1.9-A X-ray structure of the mutant showed that the carbonyl group of Asn-99 side chain is hydrogen bonded to His-48 in the same way as that of Asp-99 in the WT, thus retaining the tautomeric form of His-48 and the function of the enzyme. The NH2 group of Asn-99 points away from His-48. In contrast, in the D102N mutant of the protease enzyme trypsin, the NH, group of Asn-102 is hydrogen bonded to His-57 resulting in the inactive tautomeric form and hence the loss of enzymatic activity. Although the geometry of the catalytic triad in the PLA2 mutant remains the same as in the WT, we were surprised that the conserved structural water, linking the catalytic site with the ammonium group of Ala-1 of the interfacial site, was ejected by the proximity of the NH, group of Asn-99. The NH, group now forms a direct hydrogen bond with the carbonyl group of Ala-I.Keywords: histidine tautomeric form; missing structural water; PLA2 D99N mutant; structure-function relationship; X-ray structure Phospholipase A2 (Fig. 1A) hydrolyzes the sn-2 ester bond of phospholipids. The studies on the mechanism of action of PLA2 have generated immense pharmacological interest. The mechanism involves binding of the enzyme to the lipid-water interface, productive binding of a single lipid molecule in the active site followed by hydrolysis (Scott et al., 1990). The interfacial binding site is essentially at the surface and includes the residues of the N-terminal helix-A, the calcium ion binding loop, and the loop connecting helix-D and the 0-sheet (Dijkstra et al., 1981; see Kinemage 1). The catalytic triad Asp-99, His-48, and the waReprint requests to: Muttaiya Sundaralingam or Ming-Daw Tsai, Department

The aspartate-99 of secreted phospholipase A 2 (PLA2) has been proposed to be critical for the catalytic mechanism and interfacial activation of PLA2. Aspartate-99 connects the catalytic machinery (including the catalytic diad, the putative catalytic waters W5 and W6, and the calcium cofactor) to the hydrogen-bonding network. The latter involves Y52, Y73, the structural water, and the N-terminal region putatively required for the interfacial activation. A triple mutant of bovine pancreatic PLA2 with substitutions aspartate plus adjacent tyrosine residues (Y52,73F/D99N) was constructed, its X-ray structure was determined, and kinetic characteristics were analyzed. The kinetic properties of the D99N mutant constructed previously were also further analyzed. The X-ray structure of the Y52,73F/D99N mutant indicated a substantial disruption of the hydrogen-bonding network including the loss of the structural water similar to that seen in the structure of the D99N mutant published previously [Kumar, A., Sekharudu, Y. C., Ramakrishnan, B., Dupureur, C. M., Zhu, H., Tsai, M.-D., & Sundaralingam, M. (1994) Protein Sci. 3, 2082-2088. Kinetic analysis demonstrated that these mutants possessed considerable catalytic activity with a k cat value of about 5% compared to WT. The values of the interfacial Michaelis constant were also little perturbed (ca. 4-fold lower for D99N and marginally higher for Y52,73F/D99N). The results taken together suggest that the hydrogen-bonding network is not critically important for interfacial activation. Instead, it is the chemical step that is perturbed, though only modestly, in the mutants.Secreted phospholipases A 2 (PLA2) 1 are small calciumdependent lipolytic enzymes that catalyze the stereospecific hydrolysis of the sn-2 ester bond of phospholipids at the interfaces . The activity of PLA2 on aggregated substrates is considerably higher than on monomolecularly dispersed zwitterionic substrates. This phenomenon is known as interfacial activation. The molecular basis of interfacial activation is not known, although the hydrogenbonding network (Figure 1) has been proposed to serve as a link between the active site and the interfacial recognition site (Verheij et al., 1981;Dijkstra et al., 1983).The roles of Tyr-52, Tyr-73, and the N-terminal residues have been investigated by mutagenises (Dupureur et al., 1992a;Maliwal et al., 1994;Liu et al., 1995). The results showed that the hydrogen-bonding network involving the N-terminal region and the conserved water molecule (W11) does not play a significant role in the chemical step of the catalytic cycle at the anionic interface. The crystal structure of the double mutant Y52,73F (Sekharudu et al., 1992) showed that although the loss of hydrogen bonds of the tyrosines was compensated by the increase in the hydrophobic contacts of the phenyl groups, the structural water was retained. However, in the single mutant D99N (Kumar et al., 1994), the structural water was absent.To gain a better understanding of the role of the conserved structura...

No abstract