Mark T. Martin

An angiotensin converting enzyme inhibitor is a tight-binding slow substrate of carboxypeptidase A.

Carboxypeptidase A-catalyzed hydrolysis of peptides and depsipeptides is competitively inhibited by N-(1-carboxy-5-t-butyloxycarbonylaminopentyl)-L-phenylalanine (Boc-CA-Phe, Ki = 1.3 microM) and the angiotensin converting enzyme inhibitor, N-(1-carboxy-5-carbobenzoxyaminopentyl)-glycyl-L-phenylalanine (Z-CA-Gly-Phe, Ki = 4.5 microM). The latter compound is actually a slow substrate of carboxypeptidase. Indirect observation of inhibitor binding by stopped-flow measurement of radiationless energy transfer between carboxypeptidase tryptophans and dansylated substrates reveals slow binding for both compounds. The visible absorption spectrum of the complex of cobalt(II)-substituted carboxypeptidase and Z-CA-Gly-Phe, which differs from the corresponding spectrum of the Boc-CA-Phe complex, is remarkable in its resemblance to the spectrum of the complex between Co(II)carboxypeptidase and a transient intermediate previously observed during hydrolysis of peptide substrates. The spectrum slowly changes to that of the free enzyme indicating hydrolysis. Chromatographic quantitation of substrate and products confirms that carboxypeptidase converts Z-CA-Gly-Phe to Z-CA-Gly and L-Phe with an apparent kcat of 0.02 s-1. Absorption spectroscopy indicates that the Z-CA-Gly-Phe-Co(II)carboxypeptidase spectrum is not that of bound products. Moreover, spectral titrations indicate that the products (both with spectral Ki values of about 3 mM), as well as D-Phe, compete for the same site on the enzyme.

The catalytic mechanism of angiotensin converting enzyme and related zinc enzymes.

Angiotensin converting enzyme (ACE) is one of a class of zinc peptidases that utilize glutamic acid as a catalytic residue. ACE also has important arginine and tyrosine residues that are involved in substrate binding and a lysine that binds chloride. Chloride activation of ACE depends on the particular substrate employed. It is an essential activator of class I substrates and a nonessential activator of class II substrates. Radiationless energy transfer using substrates labeled with the fluorescent dansyl group indicates that chloride promotes a two-step binding process. The second step is a change in protein conformation that can be monitored using fluorescent inhibitors. The chloride binding constant for the ACE-inhibitor complex at pH 7.5 is 2.2 mM.

Fluorescent inhibitor probes of enzyme active site conformation: anion binding to angiotensin-converting enzyme

Dansylated tight-binding inhibitors are effective fluorophoric probes for detecting conformational changes of enzyme active sites. In this study they have been employed to examine the effect of anions on the conformation of angiotensin-converting enzyme. The efficiency of radiationless energy transfer between enzyme tryptophan residues and an active site-bound dansyl inhibitor has been shown to be enhanced by the addition of chloride. Half-maximal fluorescence enhancement occurs at about 2 mM chloride and is the same for both N-(1-carboxyl-5-dansylamino-pentyl)-glycyl-L-phenylalanine [Ki,app = 50 nM (pH 7.5, 300 mM NaCl)] and N-(1-carboxyl-5-dansylamino-pentyl)-glycyl-L-lysine (Ki,app = 5.7 nM). Other activating anions also evoke similar increases in enzyme-inhibitor energy transfer. Fluorescence changes are not due to binding additional inhibitor molecules but rather to an anion-induced change in protein conformation.

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.