1MKU

CARBOXYLIC ESTER HYDROLASE, ORTHORHOMBIC FORM OF THE TRIPLE MUTANT

Summary for 1MKU

• Entry DOI: 10.2210/pdb1mku/pdb
• Descriptor: PHOSPHOLIPASE A2, CALCIUM ION (3 entities in total)
• Functional Keywords: hydrolase, enzyme, carboxylic ester hydrolase, orthorhombic form
• Biological source: Bos taurus (cattle)
• Cellular location: Secreted: P00593
• Total number of polymer chains: 1
• Total formula weight: 13817.60

Authors

Sundaralingam, M. (deposition date: 1997-09-10, release date: 1997-12-24, Last modification date: 2024-11-13)

Primary citation

Sekar, K.,Yu, B.Z.,Rogers, J.,Lutton, J.,Liu, X.,Chen, X.,Tsai, M.D.,Jain, M.K.,Sundaralingam, M.
Phospholipase A2 engineering. Structural and functional roles of the highly conserved active site residue aspartate-99.
Biochemistry, 36:3104-3114, 1997

PubMed Abstract: 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.

PubMed: 9115986
DOI: 10.1021/bi961576x

Experimental method

X-RAY DIFFRACTION (1.8 Å)