Ionic interactions near the loop L4 are important for maintaining the active-site environment and the dimer stability of (pro)caspase 3.
We have examined the role of a salt bridge between Lys242 and Glu246 in loop L4 of procaspase 3 and of mature caspase 3, and we show that the interactions are required for stabilizing the active site. Replacing either of the residues with an alanine residue results in a complete loss of procaspase 3 activity. Although both mutants are active in the context of the mature caspase 3, the mutations result in an increase in K(m) and a decrease in kcat when compared with the wild-type caspase 3. In addition, the mutations result in an increase in the pK(a) value associated with a change in kcat with pH, but does not affect the transition observed for Km versus pH. The mutations also affect the accessibility of the active-site solvent as measured by tryptophan fluorescence emission in the presence of quenching agents and as a function of pH. We show that, as the pH is lowered, the (pro)caspase dissociates, and the mutations increase the pH-dependent instability of the dimer. Overall, the results suggest that the contacts lost in the procaspase as a result of replacing Lys242 and Glu246 are compensated partially in the mature caspase as a result of new contacts that are known to form on zymogen processing
Department of Molecular and Structural Biochemistry, 128 Polk Hall, North Carolina State University, Raleigh, NC 27695-7622, USA.
Brett
B
Feeney
Cristina
C
Pop
Ashutosh
A
Tripathy
A Clay
AC
Clark
D000409:Alanine / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D053148:Caspase 3; D020169:Caspases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D002850:Chromatography, Gel; D002942:Circular Dichroism; D019281:Dimerization; D004792:Enzyme Precursors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D011499:Protein Processing, Post-Translational; D055672:Static Electricity; D013816:Thermodynamics; D014364:Tryptophan / Q000378:metabolism
D004792:Enzyme Precursors; D014364:Tryptophan; D053148:Caspase 3; D020169:Caspases; D000409:Alanine