Allosteric Effect of Amphiphile Binding to Phospholipase A2
2009 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, no 14, 3219-3229 p.Article in journal (Refereed) Published
In the preceding paper, we showed that the formation of the second premicellar complex of pig pancreatic IB phospholipase A2 (PLA2) can be considered a proxy for interface-activated substrate binding. Here we show that this conclusion is supported by results from premicellar;E-i(#) (i = 1, 2, or 3) complexes with a wide range of mutants of PLA2. Results also show a structural bass-for the correlated functional changes during the formation of E-2(#), and this is interpreted as an allosteric T (inactive) to R (active) transition. For example, the dissociation constant K-2(#) for decylsulfate bound to E-2(#) is lower at lower pH, at higher calcium concentrations, or with an inhibitor bound to the active site. Also, the lower limits of the K-2(#) values are comparable under these conditions. The pH-dependent increase in K-2(#) with a pK(a) of 6.5 is attributed to E71 which participates in the binding of the second calcium which in turn influences the enzyme binding to phosphatidylcholine interface. Most mutants exhibited kinetic and spectroscopic behavior that is comparable to that of native PLA2 and Delta PLA2 with a deleted 62-66 loop. However, the Delta Y52L substitution mutant cannot undergo the calcium-, pH-, or interface-dependent changes. We suggest that the Y52L substitution impairs the R to T transition and also hinders the approach of the Michaelis complex to the transition state. This allosteric change may be mediated by the structural motifs that connect the D48-D99 catalytic diad, the substrate-binding slot, and the residues of the i-face. Our interpretation is that the 57-72 loop and the H48DNCY52 segment of PLA2 are involved in transmitting the effect of the cooperative amphiphile binding to the i-face as a structural change in the active site.
Place, publisher, year, edition, pages
2009. Vol. 48, no 14, 3219-3229 p.
IdentifiersURN: urn:nbn:se:uu:diva-129142DOI: 10.1021/bi801245sISI: 000264983800020OAI: oai:DiVA.org:uu-129142DiVA: diva2:337749