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.