Phospholipase A2 engineering. Structural and functional roles of the highly conserved active site residue aspartate-99.
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.
Sekar
K
K
Department of Chemistry, The Ohio State University, Columbus 43210, USA.
Yu
B Z
BZ
Rogers
J
J
Lutton
J
J
Liu
X
X
Chen
X
X
Tsai
M D
MD
Jain
M K
MK
Sundaralingam
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid; D001665:Binding Sites; D002417:Cattle; D003198:Computer Simulation; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D006860:Hydrogen Bonding; D007700:Kinetics; D008958:Models, Molecular; D010179:Pancreas / Q000201:enzymology; D010741:Phospholipases A / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D054467:Phospholipases A2; D017354:Point Mutation; D011487:Protein Conformation; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D011994:Recombinant Proteins; D001224:Aspartic Acid; D010741:Phospholipases A; D054467:Phospholipases A2