Carboxypeptidase specific inhibitors

In the case of carboxypeptidase B, Shaklai et al.(2lT> 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 iV-acetyl-L-arginine, 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. Binding of either the substrate or the inhibitor led to an increase in the emission ratio to a value similar to that of the apoenzyme. The change in the tyrosine tryptophan phosphorescence ratio was attributed to an interaction between a tyrosine and the catalytically essential zinc. The emission ratio was also studied as a function of pH. The titration data are difficult to interpret, however, because a Tris buffer was used and the ionization of Tris is strongly temperature dependent. In general, the use of Tris buffers for phosphorescence studies should be avoided. 

The discovery of teprotide led to a search for new, specific, orally active ACE inhibitors. Ondetti et al. (172) proposed a hypothetical model of the active site of ACE, based on analogy with pancreatic carboxypeptidase A, and used it to predict and design compounds that would occupy the carboxy-terminal binding site of the enzyme. Carboxyalkanoyland mer-captoalkanoyl derivatives of proline were found to act as potent, specific inhibitors of ACE and 2-D-methyl-3-mercaptopropanoyl-L-proline (131) (captopril) was developed and launched in 1981 as an orally active treatment for patients with severe or advanced hypertension. Captopril, modeled on the biologically active peptides found in the venom of the pit viper, made an important contribution to the understanding of hypertension and paved the... 

Carboxypeptidases.—A study of the biosynthesis of yeast carboxypeptidase Y (a mannoprotein) has revealed that a membrane fraction contains a precursor of the enzyme, which, although it has a higher molecular weight than the enzyme, is not a complex of the enzyme and its specific inhibitor. ... 

A cephalosporin derivative 257 with structural features of the peptidoglycan has been conceived as an inhibitor specific for DD-transpeptidases <2003JA16322>. The compound 257 has been synthesyzed in 13 steps and has been tested with recombinant PBPlb and PBP5 of E. coli, a DD-transpetidase and a DD-carboxypeptidase, respectively. It has been found that compound 257 is a time-dependent and irreversible inhibitor of PBPlb and does not interact with PBP5, neither as an inhibitor (reversible or irreversible) nor as a substrate. 

Table V) and there are carboxypeptidases that also belong to both families. Substrate specificity alone is not sufficient to rationally design an inhibitor for a new protease since it tells nothing about the active-site functional groups.

Carboxypeptidase A (approx. 35 kDa MW, Sigma Chemical) functions both as a peptidase and an esterase it is in this latter mode that it can serve as a detector for cholinesterase inhibitors. Unlike the other enzymes such as AChE or BChE, it does not have a serine residue in the active site. TPPSi forms a complex with the enzyme and, upon challenge with the cholinesterase inhibitor eserine (physostigmine) in water, exhibits a change in the absorbance spectrum with a new peak and a marked increase in absorbance at 423 nm. This suggests TPPS, may not be completely displaced from the active site. For actual sensor operations, the use of an enzyme such as BChE or carboxypeptidase in place of (or in addition to) AChE will allow for potential identification of the analyte based on different specificities/sensitivities of the enzyme. Enzymes such as OPH, which are not readily available, may be difficult to obtain in large quantities the supply of AChE is often limited perhaps due to the capture of electric eels, while proteins such as BChE (from horse blood) and carboxypeptidase (pancreas) are more readily available from slaughterhouses. 

Several peptides isolated from the venom of the South American snake Bothrops jararaca are potent ACE inhibitors and were briefly used for the treatment of hypertension, but were soon superseded by surprisingly simple molecules with high inhibitory effect. At the Squibb Institute for Medical Research in Princeton, New Jersey, Miguel A. Ondetti (Plate 32) recognized that ACE is similar in its substrate specificity to the well studied protease car-boxypeptidase A. He designed, therefore, molecules that should fit into the active site of ACE (presumably similar to the active site of carboxypeptidase A) and form complexes with the enzyme. The dipeptide , 2-D-methyl-3-mercapto-propionyl-L-proline, captopril [7] strongly associated with the enzyme and... 

However, this case is extremely rare in nature. An example is the noncompetitive inhibition of phenyllactate versus an amide substrate for carboxypeptidase. In this case, the initial collision complex of substrate and enzyme has an interaction with the terminal carboxyl and the arginine on the enzyme, as well as with the rest of the polypeptide chain, but the aromatic group of the terminal amino add is not in Ae specificity pocket. For it to seat itself requires twisting of the amide bond, which is the rate limiting and energy requiring step of the reaction. Thus, phenyllactate can slip into this pocket and prevent proper seating of the substrate. With an ester substrate, where rotation of the ester bond is not hindered, the collision complex has the specificity pocket filled, and phenyllactate is a competitive inhibitor (Auld Holmquist, 1974). 

The antinutrient properties of the proteinase inhibitor fractions have been ascribed primarily to the inhibitors of trypsin. However only limited data is available concerning the effects of inhibitors of chymotrypsin and elastase, and virtually nothing is known of the effects in animal diets of inhibitors with specificities toward the carboxypeptidase A and B. The major impediment to such research is the availability, for testing in animal diets, of inhibitors having these latter specificities. 

Inhibitors of the metallo-carboxypeptidases A and B are rare. The first such inhibitor was isolated from potatoes, and later an inhibitor with similar specificity was isolated from the intestinal worm, Ascaris lumbricoides. The latter inhibitor is difficult to purify, which precludes any nutritional studies in animals. Potato CPI, on the other hand, can represent a significant contribution to the potato proteins, depending upon variety (7). In a survey of 106 commercial and experimental varieties, CPI varied from zero to 846 yg/ml tuber juice (over 4% of the soluble proteins). In Russet Burbank potatoes the levels are about 30% of this value. The isolation of enough CPI from Russet Burbank potatoes for a nutritional study was therefore possible, if a simple efficient method could be developed to isolate it. 

Before embarking on a feeding study with CPI, it was important to establish if the inhibitor was indeed an inhibitor of chick carboxypeptidases A and B, Extracts of chick pancreata, after activation with trypsin, contained considerable carboxypeptidase A and B activities. CPI potently inhibited both chick carboxypeptidase A and carboxypeptidase B activities. The inhibition of the chick carboxypeptidases by CPI was not surprising since the inhibitor has a broad specificity toward metallo-carboxypeptidases and has been shown to inhibit all animal carboxypeptidases A and B tested to date including those of insects (9). 

Soil differed in their relative activities towards ZPL and BAA the enzymes involved were extracted from a soil with different efficiencies. Extracted ZPL-hydrolysing enzymes retained complete activity when freeze-dried, or dried at 30°C (90 min), or when incubated under bacteriostatic conditions for 10 days at 25°C. Incubation of the extracts for 1 day at 30°C with or without the addition of microbial proteinases, thermolysin or subtilisin decreased activities towards ZPL by about 10% only. The extracted enzyme, in regard to its specificity of hydrolysis of Z-dipeptides and its response to inhibitors, exhibited some of the properties of carboxypeptidase. 

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