Interaction of anions with the active site of carboxypeptidase A

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Abstract
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 in 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 displays 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. The absorption is well resolved in the corresponding MCD spectra; for example, in the Co(II)CPD .cntdot. L-Phe .cntdot. N3- complex .lambda.extremum= 323 nm and .delta..epsilon.M = + 0.207 M-1 cm-1 T-1. The absorption band is also present in thiocyanate-containing complexes; for the Co(II)CPD .cntdot. Phe .cntdot. SCN- complex .lambda.max = 316 nm and .epsilon.M 760 M-1 cm-1. MCD and EPR spectra of the ternary complexes indicate a slightly distorted tetrahedral coordination sphere around the active-site Co(II). The near-UV band in the visible and MCD spectra of a Co(II)CPD .cntdot. .beta.-phenylpropionate (.beta.-PP) .cntdot. N3- complex is lost on addition of excess .beta.-PP, suggesting that a second molecule of .beta.-PP interacts with the active-site metal ion displacing azide. The steady-state ES2 intermediate of peptide hydrolysis does not permit binding of azide to the metal, consistent with the proposal that peptides coordinate directly with the metal ion in the ES2 intermediate.