Carboxypeptidase absorptivity

Increased permeability is just one prerequisite in the development of useful peptide prodrugs. Another condition is that efficient bioactivation must follow absorption. Mucosal cell enzymes able to hydrolyze peptides include exopeptidases such as aminopeptidases and carboxypeptidases, endopepti-dases, and dipeptidases such as cytosolic nonspecific dipeptidase (EC 3.4.13.18), Pro-X dipeptidase (prolinase, EC 3.4.13.4), and X-Pro dipeptidase (prolidase, EC 3.4.13.9). For example, L-a-methyldopa-Pro was shown to be a good substrate for both the peptide transporter and prolidase. This dual affinity is not shared by all dipeptide derivatives, and, indeed, dipeptides that lack an N-terminal a-amino group are substrates for the peptide transporter but not for prolidase [29] [33] [34],... 

These proteolytic enzymes are all endopeptidases, which hydrolyse links in the middle of polypeptide chains. The products of the action of these proteolytic enzymes are a series of peptides of various sizes. These are degraded further by the action of several peptidases (exopeptidases) that remove terminal amino acids. Carboxypeptidases hydrolyse amino acids sequentially from the carboxyl end of peptides. They are secreted by the pancreas in proenzyme form and are each activated by the hydrolysis of one peptide bond, catalysed by trypsin. Aminopeptidases, which are secreted by the absorptive cells of the small intestine, hydrolyse amino acids sequentially from the amino end of peptides. In addition, dipeptidases, which are structurally associated with the glycocalyx of the entero-cytes, hydrolyse dipeptides into their component amino acids. 

The visible absorption spectrum of cobalt carboxypeptidase A was at an early stage thought to be characteristic of sulfur coordination (16, 105). The originally published spectrum (94) is poorly resolved (Fig. 14), but a more detailed study has recently been announced (106). Maxima at 555 nm (s — 160 M-1 cm-1) and 572 nm (e = 160 M-1 cm-1), a shoulder at 500 nm and a near-infrared band centered at 940 nm (e == 25 M-1 cm-1) are reported. There seems to be a clear resemblance with spectral forms... 

Fig. 14. Absorption spectra of Co(II)-carboxypeptidase A, [(CPD)Co], native carboxypeptidase A, [(CPD)Zn], and Co(H20) +, respectively. From Coleman and Valle (94)...

The most important aspect of the study of Co(II) metalloenzymes is the possibility of using the metal ion as a functional, built-in reporter of the dynamics of the active site. The spectral and magnetic properties of Co (II) carbonic anhydrase have given valuable clues to the catalytic function of this enzyme. The recent studies of Co(II) alkaline phosphatase and Co (II) carboxypeptidase A indicate the general applicability of this approach to enzymes where the probe properties of the constitutive metal ion are poor. The comparison of the absorption spectra of these enzymes and low-molecular weight models have shown that the proteins provide irregular, and in some cases nearly tetrahedral environments. It is obvious, however, that a knowledge of the crystal structures of the enzymes is necessary before the full potential of this method can be exploited. 

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. 

It is unlikely that the introduction of the probe itself induces these changes in protein conformation since recent studies with nitrocarboxy-peptidase (64) confirm the findings of Johansen and Vallee (62, 63). Treatment of carboxypeptidase crystals with tetranitromethane exclusively nitrates tyrosine-248. In solution nitrocarboxypeptidase exhibits a visible absorption band with a maximum at 428 nm titrating with a pK of about 6.3. This abnormally low pK value (relative to model nitro-... 

The accuracy of the microwave-excitation spectrometric method was verified by comparing results from it with those of atomic absorption analysis for readily available metalloenzymes of known zinc stoichiometry. Carboxypeptidase A (EC 3.4.12.2), carbonic anhydrase (EC 4.2.1.1), alcohol dehydrogenase (EC l.l.l.l), and alkaline phosphatase (EC 3.1.3.1) were dialyzed vs. metal-free bufiFers, then diluted with 10 mmol/ L KCl or 1 mmol/L HCl for metal analysis (24). For atomic absorption analysis, at least lOO- xg samples were required, but microwave excitation required only 0.1 fig. Even though 1000-fold less protein was required for microwave excitation analysis, the agreement between the data obtained by the two methods is excellent (Table II). So little of the reverse transcriptase was available to us that we could not use atomic absorption for its analyses. 

Zhang, K., Chance, B., Auld, D. S., Larsen, K. S. and Vallee, B. L. (1992) X-ray Absorption Fine Structure Study of the Active Site of Zinc and Cobalt Carboxypeptidase A in Their Solution and Crystalline... 

Figure 4-4. Intestinal absorption of dietary folates (THF-[Gluy and foUc acid (F) from fortified cereal products and vitamin supplements. In the duodenum and upper jejunum, extra glutamate residues are cleaved by conjugases (y-glutamyl carboxypeptidases). Folate (F) and reduced folate (THF) are absorbed by a proton-coupled, high affinity folate transporter into the mucosal cell, converted to N -methyl THF and exported into the portal circulation. N -methyl THF is taken up into cells by facilitative diffusion, converted to THF by the Bj -requiring methionine synthase and then converted to a polyglutamate.

Digestive enzymes are important barriers to peptide and protein absorption [3]. They are able to digest protein drugs and include trypsin, a-chymotrypsin, elastase, and carboxypeptidase A. The first three enzymes are able to cleave internal peptide bonds in many proteins (1) trypsin has more affinity for bonds near basic amino acids, such as arginine and lysine (2) a-chymotrypsin cleaves peptide linkages near hydrophobic amino acids, such as leucine, methionine, phenylalanine, tryptophan, and tyrosine (3) elastase cleaves near alanine, glycine, isoleucine, leucine, serine, and valine. Shortly after eating, about 200-800 ig of trypsin and a-chymotrypsin are present in the human duodenum. 

ABSORPTION, DISTRIBUTION, AND ELIMINATION As with vitamin Bj, the diagnosis and management of folic acid deficiency depend on an understanding of the transport pathways and intracellular metabolism of the vitamin (Figure 53-10). Folates present in food are largely in the form of reduced polyglutamates, and absorption requires transport and the action of a pteroyl-glutamyl carboxypeptidase associated with mucosal cell membranes. The mucosae of the duodenum and upper part of the jejunum are rich in dihydrofolate reductase and can methylate most or aU of the reduced folate that is absorbed. Since most absorption occurs in the proximal small intestine, it is not unusual for folate deficiency to occur with jejunal disease. Both nontropical and tropical sprues are common causes of folate deficiency. 

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