Mixed carboxypeptidase

Figure 4.19 Schematic and topological diagrams for the structure of the enzyme carboxypeptidase. The central region of the mixed p sheet contains four adjacent parallel p strands (numbers 8, 5, 3, and 4), where the strand order is reversed between strands 5 and 3. The active-site zinc atom (yellow circle) is bound to side chains in the loop regions outside the carboxy ends of these two p strands. The first part of the polypeptide chain is red, followed by green, blue, and brown. (Adapted from J. Richardson.)...

Fig. 94. Carboxypeptidase A as an example of a miscellaneous a/fi structure, (a) a-Carbon stereo, viewed from one edge of the mixed /3 sheet (b) backbone schematic, viewed as in a.

Parenthetically, it should be noted that there is evidence for the accumulation of an acylenzyme (i.e., a mixed anhydride with Glu-270) in the carboxypeptidase A-catalyzed hydrolysis of esters at low temperature, but this evidence does not include confirmation by chemical trapping experiments (Makinen et al, 1979 Kuo and Makinen, 1982 Suh et al., 1985). This would imply a nucleophilic, rather than promoted-water, pathway for ester hydrolysis. Sander and Witzel (1985) provided the only... 

One possible mechanism for the hydrolysis of peptides or esters by carboxypeptidase A involves two steps with an anhydride (acyl-enzyme) intermediate.418 In the first step, the zinc(II) activates the substrate carbonyl group towards nucleophilic attack by a glutamate residue, resulting in the production of a mixed anhydride (127). Breakdown of the anhydride intermediate is rate determining with some substrates.419 An understanding of the chemistry of metal ion effects in anhydride hydrolysis is therefore of fundamental importance in regard to the mechanism of action of the enzyme. Until recently there have been few studies of metal ion-catalysed anhydride solvolysis. 

Specific ester substrates are also hydrolyzed with carboxypeptidase A. For instance, Makinen, Fukuyama, and Kuo (27) have recently studied the enzymic hydrolysis of 0-(trans-p-ch1orocinnamoyl)-L-B-phenyl actate (CICPI.) (47),and the spin labeled nitroxide ester substrate 0-3-(2,2,5,5-tetramethylpyrrol-inyl-l-oxyl)-propen-2-oyl-L-B-phenyllactate (TEPOPL) (48). They have shown that these reactions take place via the formation of a covalent intermediate (the mixed anhydride) which can be isolated under subzero temperature conditions. The hydrolysis of CICPL and TEPOPL catalyzed by carboxypeptidase A is consequently governed by the rate-limiting breaking of the acyl-enzyme. 

Matrix-assisted laser desorption ionization is another ionization mode used for MS analysis. Enzymatically digested peptides have been studied using a 90-well microchip constmcted in a MALDI plate format (see Figure 7.41). Peptide digestion was initiated in the MALDI interface where the peptide hormone, adreno-corticotropin (ACTH) was mixed with the enzyme carboxypeptidase Y. The mixing process was self-activated in the vacuum conditions. Subsequent TOF MS analysis produced kinetic information of the peptide digestion reaction [820]. 

The ionizable ligand, EH2, is assigned to Glu270, since chemical modification of this residue with CMC (1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide) decreases the binding affinity of carboxypeptidase A for zinc and lead by more than 60- and 200-fold, respectively. A bridging interaction between the Glu270-coordinated zinc hydroxide is implicated by the ability of Zn and Pb to induce a marked increase in the visible absorption spectrum of the cobalt enzyme within 20 ms of mixing 10 " M Zn or Pb with 10 " M cobalt enzyme. 

The oscilloscope tracing in Figure 7 demonstrates the rapid enhancement of dansyl fluorescence following mixing of 1 X 10" M Dns-(Gly)3-L-OPhe and 2.5 X 10" M carboxypeptidase A and reflects the extremely rapid equilibration of enzyme and substrate to form the ES complex. The decrease in the signal is a considerably slower process and, as... 

If the a-chymotrypsin-catalysed hydrolysis of 4-nitrophenyl acetate [10] is monitored at 400 nm (to detect 4-nitrophenolate ion product) using relatively high concentrations of enzyme, the absorbance time trace is characterised by an initial burst (Fig. 5a). Obviously the initial burst cannot be instantaneous and if one uses a rapid-mixing stopped-flow spectrophotometer to study this reaction, the absorbance time trace appears as in Fig. 5b. Such observations have been reported for a number of enzymes (e.g. a-chymotrypsin [11], elastase [12], carboxypeptidase Y [13]) and interpreted in terms of an acyl-enzyme mechanism (Eqn. 7) in which the physical Michaelis complex, ES, reacts to give a covalent complex, ES (the acyl-enzyme) and one of the products (monitored here at 400 nm). This acyl-enzyme then breaks down to regenerate free enzyme and produce the other products. The dissociation constant of ES is k2 is the rate coefficient of acylation of the enzyme and A 3 is the deacylation rate coefficient. Detailed kinetic analysis of this system [11] has shown... 

Fig. 12. Enzyme tryptophan (A) and substrate dansyl (B) fluorescence during the time course of zinc carboxypeptidase catalyzed hydrolysis of DNs-Gly-L-Phe. Equal volume solutions of substrate and of enzyme, both 2.5 x 10 M, in 1 M NaCl-0.02 M Tris, pH 7.5, 25°, were mixed and the fluorescence of either tryptophan (A) or dansyl (B) was measured as a function of time under stopped-flow conditions in parallel samples, as shown by the oscilloscope tracings. Excitation was at 285 nm. Scale sensitivities for (A) and (B) are 100 mV/div. The existence of the E S complex is signalled either by (A) the suppression of enzyme tryrptophan fluorescence (quenching by the dansyl group) or (B) enhancement of the substrate dansyl group fluorescence (energy transfer from enzyme trjqrtophan). Taken from Ref. (182) with permission...

Therefore, a key argument in regard to the proposed enzymatic mechanism of carboxypeptidase A is whether the carboxylate group of Glu-270 is steri-cally capable of participating efficiently in a nucleophilic reaction. In fact, such evidence has now been obtained by spectral characterization in the subzero temperature range (—60°C) study of a covalent acyl-enzyme intermediate obtained in the hydrolysis of the specific substrate 0-(trans-p-chlo-rocinnamoyl)-L-jS-phenyllactate by carboxypeptidase A (224). Furthermore, the results indicate that deacylation of the mixed anhydride intermediate is catalyzed by a Zn-bound hydroxide group. 

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