Pancreatic hydrolysis rates

Figure 10 (Left) The rates of hydrolysis of emulsified long-chain triglycerides by pancreatic lipase. The highest rate occurs with triglycerides emulsified with gum arabic (no lecithin). All the other curves represent hydrolysis rates of triglycerides emulsified with long-chain lecithin. There is a long lag period and then a slight hydrolysis in the absence of additives. With added bile salts, the lag period is abolished, but the hydrolysis rates ate still low. Both colipase and colipase plus bile salts accelerate the hydrolytic rates. (Right) Concerted action of pancreatic lipase and phospholipase. Addition of phospholipase A2 hydrolyzes the phospholipid emulsifier (as indicated by the lysolecithin release), stops the lag phase, and initiates an accelerated triglyceride hydrolysis. (From Ref. 35.)...

Various bile salts have effects on the hydrolysis rate that do not parallel their effect on the lowering of surface tension. Thus RotUin and Schalch (303) found the order of stimulation of lipase hydrolysis was cholic acid > taurocholic acid > deoxycholic acid, but the order for maximum decrease of surface tension was deoxycholic acid > taurocholic acid > cholic acid. Kawashima (304) found that chohc acid increased the synthetic activity of pancreatic lipase to a greater extent than did deoxycholic acid, and that bile itself was far more effective than chohe acid. The special efficiency of bile was assumed to be due to the presence of amino acids. 

Products.—Considerable information concerning the mechanism of the enzymic hydrolysis of starch has been obtained from investigations of the action of purified maltase-free pancreatic amylase on a number of different substrates. The substrates studied were ordinary unfractionated but exhaustively defatted10 potato and com starches a branched chain substrate, waxy maize starch and amylose, the linear component of corn starch.41 69 eo f4 These investigations included comparisons not only of the rates of the hydrolysis of the different substrates but also of the products formed from them. 

With the same concentration of pancreatic amylase reacting under comparable conditions, no marked differences were observed in the rate of the hydrolysis of any of the unfractionated ordinary starches studied.41,69 6064 On the other hand significant differences were observed in the rate of the hydrolysis of straight and of branched chain substrates. The data60 in Table IV show that waxy maize starch is hydrolyzed more slowly than unfractionated corn starch and much more slowly than the... 

Studies of the rate of the hydrolysis of dextrins isolated from a reaction mixture after the extensive hydrolysis of starch by maltase-free malted barley alpha amylase, led Myrback11 to conclude that the flattening of the reaction curves with this amylase is not due to equilibrium between the amylase and the products of the hydrolysis. As indicated above, similar conclusions have been reached for pancreatic amylase and for the amylase of Aspergillus oryzae.41,7a... 

This example documents the difficulty of rationalizing the results of in vivo investigations when competitive metabolic reactions are seen. In such cases, simpler in vitro systems may be more informative, as exemplified by the hydrolysis of alkyl phenyl carbonates (phenyl-O-CO-O-alkyl) catalyzed by pig pancreatic elastase (EC 3.4.21.36) [15]. The rate of hydrolysis was monitored by following C02 production as with the carbamates discussed above. Indeed, the enzyme-catalyzed hydrolysis yields the phenyl hemiester of carbonic acid (phenyl-O-COOH), which decomposes rapidly to produce C02 and phenol. With these carbonates, the rate of hydrolysis decreased in the series Bu > i-Bu > Et hexyl, the /-Hu and cyclohexyl derivatives being... 

Unfortunately, the size of the crystallographic problem presented by elastase coupled with the relatively short lifedme of the acyl-enzyme indicated that higher resolution X-ray data would be difficult to obtain without use of much lower temperatures or multidetector techniques to increase the rate of data acquisition. However, it was observed that the acyl-enzyme stability was a consequence of the known kinetic parameters for elastase action on ester substrates. Hydrolysis of esters by the enzyme involves both the formation and breakdown of the covalent intermediate, and even in alcohol-water mixtures at subzero temperatures the rate-limidng step is deacylation. It is this step which is most seriously affected by temperature, allowing the acyl-enzyme to accumulate relatively rapidly at — 55°C but to break down very slowly. Amide substrates display different kinetic behavior the slow step is acylation itself. It was predicted that use of a />-nitrophenyl amid substrate would give the structure of the pre-acyl-enzyme Michaelis complex or even the putadve tetrahedral intermediate (Alber et ai, 1976), but this experiment has not yet been carried out. Instead, over the following 7 years, attention shifted to the smaller enzyme bovine pancreatic ribonuclease A. 

The investigations carried out by Professor French and his students were based on sound experimental approaches and on intuitive theoretical considerations. The latter often resulted in new experiments for testing a hypothesis. On the basis of theoretical considerations, Professor French proposed a model for the structure of the amylopectin molecule, and the distribution of the linear chains in this molecule. This model was tested by utilizing enzymes that selectively cleave the linear chains, and the results substantiated the theoretical deductions. He proposed a theory on the nature and types of reactions occurring in the formation of the enzyme - starch complex during the hydrolysis of starch by amylases. In this theory, the idea of multiple attack per single encounter of enzyme with substrate was advanced. The theory has been supported by results from several types of experiments on the hydrolysis of starch with human salivary and porcine pancreatic amylases. The rates of formation of products, and the nature of the products of the action of amylase on starch, were determined at reaction conditions of unfavorable pH, elevated temperatures, and increased viscosity. The nature of the products was found to be dramatically affected by the conditions utilized for the enzymic hydrolysis, and could be accounted for by the theory of the multiple attack per single encounter of substrate and enzyme. 

A good source of uncommon bases is tRNA. It provides substrates for studying the effect of base on the rate of hydrolysis. Baev et al. (62) showed that V2-dimethylguanylyl-(3 -5 )-cytidine-3 phosphate (G2m-pCp) was hydrolyzed much slower than the usual GpCp. Venkstern (63) reported that Tp was hydrolyzed very slowly. Naylor et al. (64) found that Cp was hydrolyzed with half the rate of CpU. The same group of workers introduced (64, 65) a chemical block on uridine and pseudo-uridine residues by reacting RNA with l-cyclohexyl-3-(2-morpho-liny]-(4)-ethyl)-carbodiimide metho-p-toluene sulfonate. The modification of the uridine residues completely blocked the action of venom exonuclease and also blocked the action of pancreatic RNase. 

Pancreatic and malt amylases gradually lose their activity in the aqueous dispersions in which they act. As above noted, there is good evidence that this is due to a destructive hydrolysis of the enzyme. The destructive action of water upon enzyme is less pronounced in the presence of substrate, probably because the combination of enzyme with substrate serves to some extent to protect the enzyme from hydrolysis. It is less rapid in solutions of commercial pancreatin and in water extracts of malt than it is in solutions of purified pancreatic and malt amylases, doubtless because of the presence in the former of substances which are products of protein hydrolysis (proteoses, peptones, polypeptids, amino acids) and whose presence therefore tends to retard further protein hydrolysis and thus to protect the enzyme protein from hydrolytic destruction, or at least to diminish the rate at which such deterioration of the enzyme occurs. 

Application and Principle This procedure is used to determine the lipase activity in preparations derived from microbial sources and animal pancreatic tissues. The assay is based on the potentiometric measurement of the rate at which the preparations will catalyze the hydrolysis of tributyrin. 

The rates of dissolution on the one hand and in vitro hydrolysis of the solid by the enzyme pancreatic lipase on the other hand are given in Fig. 7.4. If dissolution is the first step in the total hydrolysis process, the reaction scheme may be written as... 

Fig. lA Left Rates of dissolution of polymorphs A and B of CAPR Right Rates of in vitro enzymatic hydrolysis hy pancreatic hpase of polymorphs A and B of CARP. (After Andersgaard et al. 1974, with permission.)... 

Table V. Relative Maximal Rates V of Hydrolysis of Oleic Acid Esters by Pancreatic Lipase, Compared with Triolein, V = 1.0 (7)...

Figure 2. Relative rates of hydrolysis of fatty acid esters of different chain lengths hy pancreatic lipase (10)...

The action of these two pancreatic exopeptidases on synthetic substrates, proteins, and peptides has been reviewed in detail by Neurath (1960). The specificity requirements which were deduced from studies with synthetic peptides have been confirmed by studies with polypeptides. The structural requirements of specific substrates for both types of carboxy-peptidase are analogous except for the nature of the amino acids which contain the free, ionized a-carboxyl group at the terminus of the substrate. Carboxypeptidase B hydrolyzes most rapidly those bonds formed by terminal lysyl and arginyl residues, whereas carboxypeptidase A hydrolyzes terminal bonds formed by a variety of aromatic, neutral, or acidic amino acids. Of the natural amino acids only carboxyl-terminal prolyl residues are resistant to the action of the enzyme. The rate of hydrolysis depends upon the nature of the side chains of the amino acids which form the susceptible bonds. Thus, differences in the rate of hydrolysis of different substrates may vary several thousandfold. The methods for application of these peptidases to hydrolysis of proteins have been discussed in detail by Canfield and Anfinsen (1963). 

As indicated in Fig. 9.20, lipase activities were measured as a function of the orlistat surface molar fraction. With aU lipases tested, the hydrolysis of dicaprin decreased sharply as the surface molar fraction of orlistat increased. The surface molar fractions of orlistat, which reduces lipase activity to 50% (uso), are 0.013%, 0.025% and 0.25% with PPL, RGL and HGL, respectively (Fig. 9.20 A) and 0.00025% with HPL (Fig. 9.20B). On the one hand, it should be remembered that the turnover rates of gastric and pancreatic lipases are quite different [115]. Consequently, the amounts of each hpase injected into the reaction compartment of the zero-order trough were different (see legend of Fig. 9.20), leading to various orh-stat/hpase molar ratios. On the other hand, we know that monolayer systems are characterized by a low specific surface (around 1 cm /cm ). As a consequence, only a small fraction of the injected enzyme is bound to the monomolecular film. 

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