1mks
CARBOXYLIC ESTER HYDROLASE, TRIGONAL FORM OF THE TRIPLE MUTANT
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OverviewOverview
The 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.
About this StructureAbout this Structure
1MKS is a Single protein structure of sequence from Bos taurus with CA as ligand. Active as Phospholipase A(2), with EC number 3.1.1.4 Full crystallographic information is available from OCA.
ReferenceReference
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
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