Phospholipase A2 Engineering. Structural and Functional Roles of the Highly Conserved Active Site Residue Aspartate-49

Abstract

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...

On the role of water in the protein activity

“…However, despite the lack of catalytic activity, these Lys49-PLA2 homologues retain a Ca 2+ independent membrane damaging activity. In spite of the lack of catalytic activity be related in many works, some papers refute this fact and report a residual catalytic activity [39,40]. Furthermore, as such as in the case of other snake venoms, PLA2s isolated from snake venom, Lys49-PLA2s, also processes combined myotoxic and cytolytic pharmacological activities.…”

Structures of the catalytic site mutants D99A and H48Q and the calcium-loop mutant D49E of phospholipase A2

“…1a). It has been shown that Asp is involved in stabilizing the appropriate tautomer of histidine in phospholipases (Li & Tsai, 1993) and in serine proteases (e.g. Sprang et al, 1987).…”

Snake Venom Peptides: Promising Molecules with Anti-Tumor Effects

“…There are other subgroups, such as Asparagine-49, Serine-49, Glutamine-49 and Arginine -49 [76][77][78][79][80][81][82][83]. Studies have found that catalytic activity is reduced or even abolished when an Aspartic acid of native PLA2 is replaced by another amino acid [80,84].…”