1mks
CARBOXYLIC ESTER HYDROLASE, TRIGONAL FORM OF THE TRIPLE MUTANT
Structural highlights
FunctionPA21B_BOVIN PA2 catalyzes the calcium-dependent hydrolysis of the 2-acyl groups in 3-sn-phosphoglycerides. Evolutionary Conservation Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf. Publication Abstract from PubMedThe aspartate-99 of secreted phospholipase A2 (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. Phospholipase A2 engineering. Structural and functional roles of the highly conserved active site residue aspartate-99.,Sekar K, Yu BZ, Rogers J, Lutton J, Liu X, Chen X, Tsai MD, Jain MK, Sundaralingam M Biochemistry. 1997 Mar 18;36(11):3104-14. PMID:9115986[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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