Ct-Chymotrypsin

Wong and co-workers have prepared various quaternary ct-nitro-ct-methyl carboxyhc acid esters by the palladium-catalyied allyhc alkyladon of ct-nitropropionate ester fEq 5 59 The products can be kmedcaUy resolved by using ct-chymotrypsin and are converted into opdciil acdve ct-methyl ct-amino acids Such amino acids are important due to the unique biological acdvity of these nonproteinogenic ct-amino acids... 

Fig. 1 Summary of the data used to establish the complete amino acid sequence of Er-1 mating pheromone. The peptides have been designated and numbered according to the type of digest ana the theoretical order in which they appear in the sequence. Designations are CNBr, cyanogen bromide T, trypsin V8, . aureus V8 protease CT, chymotrypsin. Peptiaes indicated by two numbers connected with a hyphen result from partial cleavage. Residues directly identified by automated Edman degradation and carboxypeptidase Y digestion (CP-Y) are marked by right and left arrows, respectively, residues identified by amino acid composition are indicated by dashed lines. Taken from ref. 13 and reproduced by permission of the American Society of ...

Figure 12.5-26. Fully enzymatic synthesis of [Leujenkephalin tert-butyl ester12091. PA, penicillin acylase CT, chymotrypsin P, papain PhAc, phenyla-cetyl.

Use these, data to determine the molecular weight of ct-chymotrypsin and the value of its osmotic second virial coefficient, Bi, at these conditions. 

Lemer, J.. Lami. HL Electronic energy transfer in some class B proteins trypsin, lysozyme, ct-chymotrypsin and chymotrypsin A. In Exdted States of Biological Molecules. Birks.J. B. (Ed.) p. 601, London - New York - Toronto - Sydney Wiley 1976 Lombardi, J. R., Daffom, G. A. J. Chem. Phys. 44, 3882... 

Porcine liver esterase (PLE) gives excellent enantioselectivity with both dimethyl 3-methylglutarate [19013-37-7] (lb) and malonate (2b) diester. It is apparent from Table 1 that the enzyme s selectivity strongly depends on the size of the alkyl group in the 2-position. The hydrolysis of ethyl derivative (2c) gives the S-enantiomer with 75% ee whereas the hydrolysis of heptyl derivative (2d) results in the R-monoester with 90% ee. Chymotrypsin [9004-07-3] (CT) does not discriminate glutarates that have small substituents in the 3-position well. However, when hydroxyl is replaced by the much bulkier benzyl derivative (Ic), enantioselectivity improves significantly. 

Figure 9-6. Selective proteolysis and associated conformational changes form the active site of chymotrypsin, which includes the Aspl 02-His57-Ser195 catalytic triad. Successive proteolysis forms prochymotrypsin (pro-CT), Jt-chymotrypsin (jt-CT),and ultimately a-chymotrypsin (a-CT), an active protease whose three peptides remain associated by covalent inter-chain disulfide bonds.

To outweigh disadvantages of the kinetic resolution presented above, an enzymatic desymmetrization of prochiral sulfinyldiacetates 19 was performed. The use of various enzymes, PLE, a-chymotrypsin (a-CT) ° and PPL," made it... 

Scheme 10.1 Glycopeptide synthesis and a-chymotrypsin catalyzed release from the solid support, a) 25% TFA (CH2CI2) b) 0-(N-Boc-Phe)glycolic acid (7 eq), BOP, HOBt, DIEA b) 25% TFA (CH2CI2) c) Boc-Gly-OH (7 eq), BOP, HOBt, DIEA c) 25% TFA (CH2CI2) d) Boc-Asn(GlcNAcb)-OH (3 eq), BOP, DIEA e) galactosyl transferase, sialyl transferase f) CT, H2O, pH 7.0 g) ultrafiltration h) a-l,3-fuco-syltransferase, GDP-Fuc (2.5 eq), 0.1 M HEPES (pH 7.0), 95%.

Scheme 10.2 Oligosaccharide synthesis and a-chymotrypsin catalyzed release from the solid support, a) Z-Phe-NH-(CH2)6-OH (7), CSA, (CHCljjz, 70°C b) Hj, Pd/C, MeOH, 50°C c) CH2 = CHCONH(CHjjsCOOH (8), EtOH-CeHe d) MeONa (cat.), MeOH/THF, CH2 = CHCONH2, TMEDA, APS, DMSO-H2O, SOT e) Galactosyl transferase, Sialyl transferase g) CT, Tris-HCI buffer, pH 7.8, 48 °C, 72% from (10).

Figure 16.13 The free energy of denaturation AfjG as a function of temperature for a number of proteins Lys = lysozyme Rna = ribonuclease A Ct = a-chymotrypsin Cyt = cytochrome c Mb = metmyoglobin Tr = Trypsin and PTI2 = the dimer of pancreatic trypsin inhibitor. Reprinted with permission from P. L. Privalov, Stability of Proteins — Small Globular Proteins, Adv. Prot. Chem., 33, 167 (1979).

For a-chymotrypsin, the procedure of active-site titration for the calculation of active enzyme concentration and thus of the catalytic constant kcat is long established. The original active-site titration experiment on a-CT by Hartley and Kilby (Hartley, 1954) was performed with ethyl p-nitrobenzoate (Figure 9.2). 

Proteins such as cytochrome c (Cyt c), met-myoglobn (Mb), met-hemoglobin (Hb), lysozyme (Lys), glucose oxidase (GO), and a-chymotrypsin (CT) bind to the galleries of a-ZrP (at room temperature and pH 7.2) and these proteins also retained their activities and structures upon binding [99],... 

In terms of structure and function, the 265 proteasome is an ATP-dependent multicatalytic enzyme complex comprising one or two 195 regulatory caps and a proteolytic 205 core particle within which protein degradation occurs.18,25,26 The 205 proteasome contains three pairs of proteolytic subunits, (35, (32 and (31, for which chymotrypsin-like (CT-L), trypsin-like (T-L) and caspase-like (C-L) activities have been ascribed, respectively, based on their substrate preferences.27... 

We report the results from a molecular dynamics simulation of the serine protease y-chymotrypsin (y-CT) in hexane. The active site of chymotrypsin contains the "catalytic triad" which consists of Ser-His-Asp. y-CT suspended in nearly anhydrous solvents has been found to be catalytically active. In order for proteins to retain their activity in anhydrous solvents some water molecules are required to be present. These "essential waters" have been suggested to function as a molecular lubricant for the protein. Hexane, having a dielectric constant of 1.89, is a suitable non-aqueous solvent for enzymatic reactions. The low dielectric constant of hexane allows it to not compete with the protein for the essential water and allows enzymes to retain their catalytic activity. y-CT in hexane is thus an ideal system to further explore the effect of non-aqueous solvation on protein structure, function and dynamics. 

Challenge Problem. The hydrolysis of N-glutaryl-L-phenylalanine-p-nitroanilide (GPNA) by the enzyme a-chymotrypsin (CT) to form p-nitroaniline and A-glutaryl-L-phenylalanine follows the Michaelis-Menten mechanism in its early. stages. 

Fig. 1. Rapid-scanning stoppcd-flow (RSSF) study of the reaction ofN-furylacryloylr-tryptophan methyl ester (FATME) with a-chymotrypsin (a-Ct) at pH 5.0 in the absence and presence of proflavin. (A) RSSF difference spectra for the reaction of 19 pM a-Ctwith 7.5 pM FATME in 0.1 M pH 5.0 sodium acetate buffer at 25°. Spectrum 0 is 7.5 pM free FATME, spectra 1 -5 are difference spectra measured during reaction wherein the spectrum of a-Ct has been subtracted from the set. Spectrum 6 is the spectrum of the hydrolysis product furylacryloyl i-tryptophan with the spectrum of a-Ct removed by subtraction. Spectra were measured at the following time intervals after flow had stopped (1) 8.54, (2) 162.3, (3) 341.6 (4) 1409.1 and (5) 3074.4 ms. Spectrum 6, t = oo.

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