Effects of mechanism-based reversible inhibitors on the metal environment of cobalt(II)carboxypeptidase A: an electronic spectral study.
Electronic absorption, circular dichroic (CD), and magnetic circular dichroic (MCD) spectra have been determined for complexes of cobalt(II)-substituted carboxypeptidase A and five reversible inhibitors. Three of the inhibitors, N-(1-carboxy-5-butyloxycarbonylaminopentyl)-L-phenylalanine, (I); (R,S)-2-benzyl-4-oxobutanoic acid, (III); and 2-benzyl-4-oxo-5,5,5-trifluoropentanoic acid, (IV) are mechanism-based inhibitors. Another, N-(1-carboxy-5-carbobenzoxyaminopentyl)-glycyl-L-phenylalanine, (II), is a tight binding, slowly hydrolyzed substrate. The fifth, phosphoramidon, (V), is a mechanism-based inhibitor of thermolysin, and may also bind to carboxypeptidase in a mechanism-based mode. The absorption and CD spectra of the enzyme-inhibitor complexes all differ from the spectrum of the free enzyme and from each other. The MCD spectra indicate that the tetrahedral coordination geometry of cobalt, which is distorted in the free enzyme, is also distorted in the inhibitor complexes, although to various degrees. The complexes of I and III are spectrally similar despite being structurally dissimilar, and that of IV, whose structure resembles III, is spectrally distinct, indicating that I and III, but not IV, may perturb the metal in nearly the same way. The absorption spectrum of IV is identical to that, at high pH, of Co(II)carboxypeptidase in which Glu-270 has been modified by a carbodiimide reagent, possibly pointing to a common perturbation of this residue. The absorption and CD spectra of II are similar to those of the catalytic intermediate that precedes the rate-limiting step in peptide hydrolysis [D. S. Auld, A. Galdes, K. F. Geoghegan, B. Holmquist, R. Martinelli, and B. L. Vallee, Proc. Natl. Acad. Sci. USA 81, 4675-4681 (1984)]. Since II is a substrate, the steady-state bound species that it generates may therefore be a true productive intermediate rather than a nonproductive mimic of an intermediate. The spectra of the complexes with II and V differ considerably despite structural similarities. The negative CD ellipticity of the free enzyme is reversed in sign in the presence of V, a phenomenon previously observed with complexes of Co(II)carboxypeptidase and dipeptides. This resemblance may result from a similar interaction of cobalt with the phosphoramidate group of phosphoramidon and the N-terminal amine of dipeptides. The spectra of reversible, mechanism-based inhibitors permit general structural predictions about true intermediates but require caution when used for assigning precise conformation and ligands of bound catalytic species.
Martin
M T
MT
Center for Biochemical and Biophysical Sciences and Medicine, Harvard Medical School, Boston, MA 02115.
Holmquist
B
B
Riordan
J F
JF
D001665:Binding Sites; D002268:Carboxypeptidases / Q000037:antagonists & inhibitors*; D043422:Carboxypeptidases A; D002942:Circular Dichroism; D003035:Cobalt; D013053:Spectrophotometry
D003035:Cobalt; D002268:Carboxypeptidases; D043422:Carboxypeptidases A