Interaction of anions with the active site of carboxypeptidase A.
Studies of azide inhibition of peptide hydrolysis catalyzed by cobalt(II) carboxypeptidase A identify two anion binding sites. Azide binding to the first site (KI = 35 mM) inhibits peptide hydrolysis in a partial competitive mode while binding at the second site (KI = 1.5 M) results in competitive inhibition. The cobalt electronic absorption spectrum is insensitive to azide binding at the first site but shows marked changes upon azide binding to the second site. Thus, azide elicits a spectral change with new lambda max (epsilon M) values of 590 (330) and 540 nm (190) and a KD of 1.4 M, equal to the second kinetic KI value for the cobalt enzyme, indicating that anion binding at the weaker site involves an interaction with the active-site metal. Remarkably, in the presence of the C-terminal products of peptide or ester hydrolysis or carboxylate inhibitor analogues, anion (e.g., azide, cyanate, and thiocyanate) binding is strongly synergistic; thus, KD for azide decreases to 4 mM in the presence of L-phenylalanine. These ternary complexes have characteristic absorption, CD, MCD, and EPR spectra. The absorption spectra of azide/carboxylate inhibitor ternary complexes with Co(II)CPD display a near-UV band between 305 and 310 nm with epsilon M values around 900-1250 M-1 cm-1. The lambda max values are close to the those of the charge-transfer band of an aquo Co(II)-azide complex (310 nm), consistent with the presence of a metal azide bond in the enzyme complex.(ABSTRACT TRUNCATED AT 250 WORDS)
Bicknell
R
R
Center for Biochemical and Biophysical Sciences and Medicine, Harvard Medical School, Boston.
Schäffer
A
A
Bertini
I
I
Luchinat
C
C
Vallee
B L
BL
Auld
D S
DS
D000838:Anions; D001386:Azides / Q000494:pharmacology; D001665:Binding Sites; D002268:Carboxypeptidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D043422:Carboxypeptidases A; D002942:Circular Dichroism; D003035:Cobalt / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D011485:Protein Binding; D015032:Zinc / Q000378:metabolism
D000838:Anions; D001386:Azides; D003035:Cobalt; D002268:Carboxypeptidases; D043422:Carboxypeptidases A; D015032:Zinc