The Role of a Tyrosyl Residue in the Mechanism of Action of Carboxypeptidase B: Luminescence Studies

Abstract

The luminescence spectra of carboxypeptidase B indicate specific differences between the zinc and apoenzyme due to the state of tyrosyl residues presumably at the active site. These differences disappear when enzyme-substrate or enzyme-inhibitor co6mplexes are formed, suggesting that they may reflect the interaction of a tyrosyl residue in the native enzyme with the catalytically essential zinc atom. An interpretation of the role of that tyrosyl residue in the mechanism of action of carboxypeptidase B is presented.Chemical modifications have implicated a chemically hyperreactive tyrosyl residue in the function of carboxypeptidase B. Thus, nitration of the enzyme (1) markedly decreases the enzymic activity towards both basic and nonbasic substrates (2). These changes have been correlated with the nitration of active-site tyrosyl residues. Alkylation (3), iodination (5), acetylation (4), succinylation (6), and coupling with a diazonium salt (6) also involve tyrosyl residues with analogous catalytic consequences. The luminescence spectra of proteins provides an intrinsic sensitive signal of changes in stereochemical relationships directly related to the aromatic aminoacid side chains, especially tryptophan and tyrosine. Changes in the microenvironment of these residues may be thus observed especially in a protein like carboxypeptidase B possessing a reactive tyrosyl residue. We have, therefore, examined the luminescence properties of this enzyme. The results indicate differences in the tyrosyl state between the apo-and the zinc enzyme. These differences disappear on formation of enzyme-substrate or enzyme-inhibitor complexes. METHODSApocarboxypeptidase B was prepared from porcine carboxypeptidase B [(Code COBC) Worthington Biochemical Corp.] by dialysis against multiple changes of 4 mM 1,10-phenanthroline in 0.01 M sodium acetate buffer (pH 5.5) followed by dialysis against the same buffer without phenanthroline, and finally against 0.05 M Tris HCl-0.1 M NaCl at the desired pH. The apoenzyme was reconstituted by dialysis against 0.05 M Tris HCl-0.1 M NaCl (pH 7.5) buffer containing 1 mM zinc sulphate (Merck Co.) followed by dialysis against metal-free buffer.The metal content was determined by atomic absorption spectroscopy (Varian Techtron model AA5) and found to be 0.96-0.98 g-atom of Zn in the reconstituted enzyme and less than 0.03 g-atom of Zn in the apoenzyme. Enzymatic * To whom to address correspondence. activities were estimated with basic and nonbasic peptide and ester substrates (2).Fluorescence spectra were measured with a HitachiPerkin-Elmer spectrofluorometer model MPF-2. Phosphorescence measurements were performed with the same instrument, equipped with a phosphorescence attachment. Phosphorescence signals were obtained by use of a chopper of the Becquerell type rotating at 25 Hz to cut off short-lived signals (fluorescence and stray-light). Emission was observed at 900 to the incident beam. All spectra given are uncorrected for instrument response. Decay curves were obtained by use of a Te...

The Physical State Dependence of Carboxypeptidase A α and A γ Kinetics

“…In this context it is remarkable that in the face of the accumulating experimental evidence for conformational (11)(12)(13)17) and functional (14)(15)(16) differences between crystal shown to be functions of the physical state, though the basis of these differences could not then be ascertained (13)(14)(15).…”

“…Thus, either nitration of Tyr-248 or coupling it with diazoarsanilic acid reveals that, in solution, the location of this residue with respect to the zinc atom differs from that in the crystals (11)(12)(13). Moreover, even prior to the x-ray structure determination, the activity of enzyme crystals toward Cbz Gly-Phe was found to be much lower than that of its solutions, strongly suggesting the possibility of different conformations in the two physical states (14)(15)(16) (11)(12)(13)17). These kinetic data constitute, in fact, telling evidence of the functional consequences of such conformational differences, thereby making available novel indices for their detection.…”

Tyrosine Fluorescence and Phosphorescence from Proteins and Polypeptides

“…(202) The same approach has been used to study the role of tyrosine in the mechanism of action of carboxypeptidase B. (217,218) In both these proteins, as in other proteins which contain both tyrosine and tryptophan, the tyrosine fluorescence is difficult to resolve from the tryptophan fluorescence. The tyrosine phosphorescence, however, is better resolved from the tryptophan phosphorescence since the high-energy edges of their emission bands are separated by about 50 nm; the high-energy edge of tyrosine phosphorescence begins near 350 nm whereas that of tryptophan typically begins near 400 nm.…”

“…In the case of carboxypeptidase B, Shaklai et al (217) compared the relative contributions to the protein phosphorescence from tyrosine and tryptophan for the apoenzyme, the zinc-containing metalloenzyme in the absence of substrate, the metalloenzyme in the presence of the substrate N-acetyl-Larginine, and the metalloenzyme in the presence of the specific inhibitor L-arginine. The tyrosine : tryptophan emission ratio of the metalloenzyme was about a factor of four smaller than that of the apoenzyme.…”