Control of the chemical step by leucine-31 of pancreatic phospholipase A(2)

Citation
Bz. Yu et al., Control of the chemical step by leucine-31 of pancreatic phospholipase A(2), BIOCHEM, 39(19), 2000, pp. 5702-5711
Citations number
45
Categorie Soggetti
Biochemistry & Biophysics
Journal title
BIOCHEMISTRY
ISSN journal
00062960 → ACNP
Volume
39
Issue
19
Year of publication
2000
Pages
5702 - 5711
Database
ISI
SICI code
0006-2960(20000516)39:19<5702:COTCSB>2.0.ZU;2-T
Abstract
A well-defined region of pancreatic and other secreted phospholipase A(2) ( PLA2), which we call the i-face, makes a molecular contact with the interfa ce to facilitate and control the events and processivity of the interfacial catalytic turnover cycles. The structural features of the i-face and its a llosteric relationship to the active site remain to be identified. As a par t of the calcium binding (26-34) loop, Leu-31 is located on the surface nea r the substrate binding slot of PLA2. Analysis of the primary rate and equi librium parameters of the Leu-31 substitution mutants of the pig pancreatic PLA2 shows that the only significant effect of the substitution is to impa ir the chemical step at the zwitterionic interface in the presence of added NaCl, and only a modest effect is seen on k*(cat) at the anionic interface . Leu-31 substitutions have Little effect on the binding of the enzyme to t he interface; the affinity for certain substrate mimics is modestly influen ced in W3F,L31W double mutant. The fluorescence emission results with the d ouble mutant show that the microenvironment of Trp-31 is qualitatively diff erent at the zwitterionic versus anionic interfaces. At both of the interfa ces Trp-31 is not shielded from the bulk aqueous environment as it remains readily accessible to acrylamide and water. The NaCl-induced change in the Trp-31 emission spectrum of the double mutant on the zwitterionic interface is similar to that seen on the binding to the anionic interface. Together, the kinetic and spectroscopic results show that the form of PLA2 at the zw itterionic interface (E-z*) is distinguishably different from the catalytic ally more efficient form at the anionic interface (E-a*). This finding prov ides a structural basis for the two-state model for k*(cat) activation by t he anionic interface. In conjunction with earlier results we suggest that n eutralization of certain cationic residues of PLA2 exerts a control on the calcium loop through residue 31.