Chymotrypsin substrates

Benzamide known as benti romide, is a chymotrypsin substrate of value as a diagnostic acid for assessment of pancreatic function. It is synthesized by amide formation between... 

Besides this problem of designing conformationally restricted analogs for highly specific enzymes, there are Other problems to be considered in dealing with less specific enzymes. These are discussed later in the section on locked a-chymotrypsin substrates. 

The facile reaction of CAA and BAA with nucleosides and nucleotides is one example of many of the applications of the bifunctional reactivity of halogenated aldehydes and ketones in modification of biomolecules. In an early example of the extensive use of halogenated ketones as protease substrate analogues, l-V-tosylamido-2-phenylethyl chloro-methyl ketone (TPCK) 30 was synthesized as a chymotrypsin substrate analogue. Stoichiometric inhibition was accompanied by loss of one histidine residue as a result of alkylation by the chloromethyl moiety68. A host of similar analogues were subsequently prepared and used as selective enzyme inhibitors, in particular for the identification of amino acid residues located at enzyme active sites69. 

Products on hydrolysis for 24 hr at pH 9, 30°C, wdth a chymotrypsin substrate molar ratio of 1 150... 

Processes. III. The Inhibition of Platelet Aggregation by Chymotrypsin Substrates and Inhibitors, Thrombos, Diathes, Haemorrh. (1970) 23,... 

A peptide (73 residues) which has chymotrypsin-like esterase activity has been designed and synthesized by Hahn et al. (1990). The peptide consists of a bundle of four short helical sequences, and the catalytic residues were synthesized at the amino end of the bundle in spatial relationships similar to chymotrypsin. Also, the oxyanion hole and substrate binding pocket for acetyltyrosine ethyl ester, a chymotrypsin substrate, have been included in the design, the peptide cleaves this ester at a rate that is 1/100 of the chymotrypsin rate. It is also inhibited by chymotrypsin inhibitors. 

M. Norin, A. Mattson, T. Norin, and K. Huh, Biocatalysis, 7, 131 (1993). Molecular Modelling of Chymotrypsin-Substrate Interaaions Calculation of Enantioselectivity. 

It was recognized some time ago that the binding and catalysis of a-chymotrypsin substrates correlated with their hydrophobicity [32] and subsequent X-ray studies... 

Figure 5. Activity-pH curves. Key O, chymotrypsin , a polyanionic ethylene-maleic acid (EM A) copolymer derivative of chymotrypsin, and A> a polycat ionic derivative, polyornithyl chymotrypsin. Substrate is acetyl-L-tryrosine ethyl ester. Ionic strength is 0.01 mol/L. Reproduced, with permission, from Ref. 19. Copyright 1976, Academic Press, Inc.

Figure 14.4.3.2. The dependence of (A) subtilisin Carlsberg and (B) a-chymotrypsin substrate specificity for substrates 1 and 2 on the ratio of their Raoult s law activity coefficients. For the stractures of the substrates 1 and 2, and the solvents a through m in (A) and a to g in (B), refer to Ref 16. [Adapted, by permission, Ifom C.R. Wescott and A.M. Klibanov, Biotechnol. Bioeng., 56, 343(1997)].

Elastase (EC 3.4.21.11) an endopeptidase specific for the Elastic (see) in animal elastic fibers. Its inactive precursor, proelastase, is formed in the vertebrate pancreas and converted in the duodenum to elastase by the action of trypsin. The natural substrate of E. is elas-tin, an insoluble protein rich in valine, leucine and isoleucine. E. attacks the peptide bond adjacent to a nonaromatic, hydrophobic amino acid. The best synthetic substrates are therefore acetyl-Ala-Ala-Ala-OCHj and benzoylalanine methyl ester. Benzoylarginine ester (a trypsin substrate), and acetyltyrosine ester (a chymotrypsin substrate) are not attacked by E. 

Also important is the finding that not only the conformer is inert to hydrolysis by a-chymotrypsin, but it also failed to inhibit enzymatic hydrolysis of the active S,Seq conformer. In marked contrast, l-KCTI has been shown to strongly inhibit chymotryptic hydrolysis of d-KCTI. This pattern of competitive inhibition has also been demonstrated for other enantiomeric pairs of chymotrypsin substrates. To understand this behavior it should be realized that the two conformers of Belleau s compound differ in two important aspects orientation of the carbomethoxyl group and the chirality of the biphenyl system. Consequently, it must be concluded that in its reaction with this constrained substrate, a-chymotrypsin displays specific recognition of molecular asymmetry. This is referred to as tertiary structural specificity. The specificity of the biphenyl compound thus serves to extend the concept that appropriately constrained substrates can serve as very useful tools. 

The sidechain of methionine-192 plays an important role in a-chymotrypsin, being implicated in both the activation of the enzyme from its zymogen and the binding and orientation of a-chymotrypsin substrates during catalysis. X-ray diffraction studies reveal that the sidechain may act as a lid over a hydrophobic sidechain binding pocket known as the tosyl hole. Modification of the sidechain of Met-192 therefore has kinetic consequences thus the addition of an aromatic moiety allows it to bind in the tosyl pocket and to compete for this site with substrate or inhibitor. The behavior of such an aromatic moiety may conveniently be followed via... 

---