Catalytic conformation of carboxypeptidase A. Structure of a true enzyme reaction intermediate determined by electron nuclear double resonance.
The structure of a catalytically competent reaction intermediate of carboxypeptidase A (CPA) formed with the specific spin-label ester substrate O-[3-(2,2,-5,5-tetramethyl-1-oxypyrrolinyl)propen-2-oyl]-L-b eta- phenyllactate through application of cryoenzymological methods has been determined by electron nuclear double resonance (ENDOR) and molecular modeling. It is shown that the reaction intermediate is best identified as a mixed-anhydride acylenzyme derivative in which the side chain of Glu-270 is acylated by the spin-label substrate, in agreement with previous cryoenzymological and spectroscopic studies from this laboratory. From the observed proton ENDOR shifts corresponding to principal hyperfine coupling components and assigned by selective deuteration, the dipolar hyperfine coupling components were calculated to estimate electron-proton distances. With these ENDOR-determined distances as constraints, the conformation of the substrate free in solution and in the active site of CPA has been determined on the basis of torsion angle search calculations. With a catalytically active, acetylated form of CPA, we have also assigned the position of the side chain of Tyr-198 with respect to the nitroxyl group. The positional assignments of both substrate and active-site residues in a true reaction intermediate provide important constraints in defining the structural basis of action of CPA.
Mustafi
D
D
Department of Biochemistry and Molecular Biology, University of Chicago, Illinois 60637.
Makinen
M W
MW
D001665:Binding Sites; D002268:Carboxypeptidases / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000648:ultrastructure*; D043422:Carboxypeptidases A; D002384:Catalysis; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013113:Spin Labels; D013386:Succinates / Q000494:pharmacology
D010455:Peptides; D013113:Spin Labels; D013386:Succinates; C030196:benzylsuccinate; D002268:Carboxypeptidases; D043422:Carboxypeptidases A