Chymotrypsin activation volumes

Figure 4 shows the temperature profiles for amidase activity of the protease from M. jannaschii at 10,250, and 500 atm. The optimum temperature for activity of 116° at low pressure (10 atm) is one of the highest optimum temperatures for a proteolytic enzyme reported in the literature. Figure 4 also shows that pressure substantially enhances the activity of the protease at each temperature for example, application of 500 atm increases the maximum reaction rate about twofold and the rate at 130°C fivefold. The 400% enhancement of amidase activity at 130° translates to an overall activation volume, A V, of — 106 ml mol , as determined by the Johnson-Eyring equation [Eq. (2)]. The most negative activation volumes previously reported for serine proteases were —36 ml mol for the digestion of casein by trypsin and —33 ml mol for the hydrolysis of Suc-Ala-Ala-pNA by a-chymotrypsin. Michels and Clark also found that the protease is stabilized... 

As mentioned in Volume 13 of these Reports, 4-oxoazetidin-2-yl phosphonates and phosphinates (19) can be prepared by Arbusov-like reactions between P compounds and 4-acetoxyazetidin-2-one (20). Acid hydrolysis of (19) yields phosphono- and phosphino-aspartic acids (21) which can be converted into peptides with antibacterial activity. Diastereomeric mixtures of phosphono-dipeptides, which can be prepared from racemic dialkyl 1 -aminoalkylphosphonates, can be separated by ion-exchange chromatography. It appears that it is easier to synthesize phosphonodipeptides from these phosphonates as their P-dialkyl esters rather than as the free phosphonic acids. Phosphonic acid analogues of A-Cbz-alanine and -phenylalanine can be converted into ester and amide fluoridates, e.g., (22, R = OMe or NHCHMeg). These fluoridates are the most potent inhibitors of elastase and chymotrypsin yet reported and seem to mimic the natural substrates of these enzymes. ... 

FIG. 7 Dependence on the water/surfactant molar ratio of (a) the maximal reaction rate normalized to the enzyme concentration, V/E, of a-chymotrypsin-catalyzed hydrolysis of N-benzoyl-L-tyrosine p-nitroanilide, and (b) the rotational frequency, v, of the spin label in the active site of the enzyme in the system AOT-water/glycerol-octane. Water/glycerol volume ratios 1—100 0 2—80 20, 3—50 50, 4—20 80, 5—6 94. Dashed lines show V/Eq and v values in aqueous solution. (From Ref. 42.)... 

Presented in Fig. 8 are the data on the determination of the minimal amount of water required for a-chymotrypsin catalysis. The system CTAB-dimethyl sulfoxide/water-octane/chloroform was used as a reaction medium, and the volume ratio of water to dimethyl sulfoxide was varied from 0 to 0.001 [12,44]. It can be seen that in the totally dry system the reaction does not take place. Introduction of water activates the enzyme. Its full activation (defined by titration) occurs in the presence of just a few molecules of water (around 5) per enzyme molecule. The plateau in Fig. 8 is explained by the fact that the enzyme used is a hydrolase in low-water conditions acylation of the enzyme by the substrate occurs with quantitative formation of the acyl-enzyme. (Incidentally, it is a direct demonstration of acyl-enzyme formation in the reaction of chymotrypsin with an anilide substrate.) Hydrolysis of acyl-enzyme by water occurs at higher water concentrations, when, apparently, water appears as a reagent in a free state. 

FIG. 11 Combination of two factors regulating enzyme catalytic activity variation in the surfactant concentration and addition of water-miscible organic solvents, (a) Peroxidase in the system AOT-water/glycerol-octane at water/glycerol volume ratios (O) 100 0 ( ) 20 80. (b) a-Chymotrypsin in the system AOT-water/glycerol-octane at water/glycerol volume ratios (O) 100 0 ( ) 6 94. Dashed lines show the catal5dic activities of the enzymes in aqueous solution. (From Ref. 44.)... 

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