Desolvation map of the i-face of phospholipase A2.
The changes in the microenvironment of the Trp-3 on the i-face of pig pancreatic IB phospholipase A2 (PLA2) provide a measure of the tight contact (Ramirez and Jain, Protein Sci. 9, 229-239, 1991) with the substrate interface during the processive interfacial turnover. Spectral changes from the single Trp-substituent at position 1, 2, 6, 10, 19, 20, 31, 53, 56 or 87 on the surface of W3F PLA2 are used to probe the Trp-environment. Based on our current understanding only the residue 87 is away from i-face, therefore all other mutants are well suited to report modest differences along the i-face. All Trp-mutants bind tightly to anionic vesicles. Only those with Trp at 1, 2 or 3 near the rim of the active site on the i-face cause significant perturbation of the catalytic functions. Most other Trp-mutants showed < 3-fold change in the interfacial processive turnover rate and the competitive inhibition by MJ33. Binding of calcium to the enzyme in the aqueous phase had modest effect on the Trp-emission intensity. However, on the binding of the enzyme to the interface the fluorescence change is large, and the rate of oxidation of the Trp-substituent with N-bromosuccinimide depends on the location of the Trp-substituent. These results show that the solvation environment of the Trp-substituents on the i-face is shielded in the enzyme bound to the interface. Additional changes are noticeable if the active site of the bound enzyme is also occupied, however, the catalytically inert zymogen of PLA2 (proPLA2) does not show such changes. Significance of these results in relation to the changes in the solvent accessibility and desolvation of the i-face of PLA2 at the interface is discussed.
Tsai
Yu-Cheng
YC
Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, USA.
Yu
Bao-Zhu
BZ
Wang
Yu-Zhen
YZ
Chen
Junghuei
J
Jain
Mahendra K
MK
D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D017931:DNA Primers; D008958:Models, Molecular; D010084:Oxidation-Reduction; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D012995:Solubility; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013552:Swine
D017931:DNA Primers; D010741:Phospholipases A; D054467:Phospholipases A2