Chymotrypsin difference spectra

Many enzymes have absolute specificity for a substrate and will not attack the molecules with common structural features. The enzyme aspartase, found in many plants and bacteria, is such an enzyme [57], It catalyzes the formation of L-aspartate by reversible addition of ammonia to the double bond of fumaric acid. Aspartase, however, does not take part in the addition of ammonia to any other unsaturated acid requiring specific optical and geometrical characteristics. At the other end of the spectrum are enzymes which do not have specificity for a given substrate and act on many molecules with similar structural characteristics. A good example is the enzyme chymotrypsin, which catalyzes hydrolysis of many different peptides or polypeptides as well as amides and esters. 

Detection of the intermediate is possible if it has a spectrum sufficiently different from that of the enzyme. The cinnamoyl chymotrypsin intermediate is characterised by a UV maximum at 292 nm the acyl papain intermediate JV-benzoylaminothionacetyl papain has a UV maximum at 313 nm. The UV absorptions of the reactions catalysed by papain and chymotrypsin wax and wane in the presence of substrate giving rise to these intermediates. 

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.

Figure 1 shows a composite of the F NMR spectra (measured on a Bruker HFX-90 at 84.669 Ifflz) of o-CF labeled a-chymotrypsin a) at pH 4.2, b) in the presence of 8.5 mM indole, and c) in 6 M urea. Note the minor narrower peak slightly upfield from the naj ve peak in a) and b), respectively, which was found to represent f labeled enzyme which was subsequently autolyzed. While this in5>urity peak was removable by CM-52 chromatography, a sample left in solution for several days developed this additional autolysis peak which was always distinctly different from the urea denatured form of the enzyme, spectrum c). Similar spectra were observed for the m CF and p-CF labeled derivatives as well. Only the native peak was sensitive to indole binding as exemplified in Figure la-b. 

Figure 4-37. Subtractive HODS, la fundamental spectrum of RNase (cone. 0.250 gL in vi ater) lb third derivative of la 2a fundamental spectrum of chymotrypsin (cone. 0.125 g L in water) 2b third derivative of la 3a mixture of RNase and chymotrypsin (cone. 0.375 g L enzymes) 3b third derivative of 3a 4a computed subtraction of 3a-la- 4a (almost identical with 2a) 4b computed subtraction of 3b-lb- 4b. The d spectra 2b and 4b show only small differences MD D (PP) [10, 29].

---