N- Acetyl -L-phenylalanine p-nitrophenyl ester

A study of the enzyme s kinetics provided a second clue to chymotrypsin s catalytic mechanism. The kinetics of enzyme action are often easily monitored by having the enzyme act on a substrate analog that forms a colored product. For chymotrypsin, such a chromogenic substrate is N-acetyl-L-phenylalanine p-nitrophenyl ester. This substrate is an ester rather than an amide, but many proteases will also hydrolyze esters. One of the products formed by chymotrypsin s cleavage of this substrate is p-nitrophenolate, which has a yellow color (Figure 9.3). Measurements of the absorbance of light revealed the amount of p-nitrophenolate being produced. 

N-Acetyl-L-phenylalanine p-nitrophenyl ester yields a yellow product, p-niUophenolate, on cleavage by chymotrypsin. p-Nitrophenolate forms by deprotonation of p-nitrophenol at pH 7. 

Now you see it, now you don t. Pre-steady-state experiments using chymotrypsin and a chromogenic substrate (N-acetyl-L-phenylalanine p-nitrophenyl ester) show a burst of product at very short times (Figure 9.4). The Conceptual Insights module on enzyme kinetics explains this result. What results would you see if the product detected by the assay was the free N-terminal component of the substrate instead of the C-terminal component Hint Use the pre-steady-state reaction simulation to simulate the experiment. Select different times following mixing and observe the amount of each product). 

The answer concerns the different kinetic behavior of chymotrypsin toward amide and ester substrates. Substrate A is N-acetyf-L-phenyfalanine p-nitrophenyl amide, rather than N-acetyl-L-phenylalanine p-nitrophenyf ester for which the initial burst activity was described in the text. The burst is observed if the first step of a reaction (in this case, acyl-chymotrypsin formation, together with release of p-nitrophenyl amine) is much faster than the second step (release of N-acetyl-phenylalanine and free chymotrypsin). With the amide substrate, however, the relative rates of the two steps are more nearly equal therefore no burst is observed. 

Cyclobis(N-methyl-L-phenylalanyl), 42B, 358 N-Acetyl L phenylalanyl-L-tyrosine, 39B, 365 Carbobenzoxy-L-leucyl-p-nitrophenyl ester, 40B, 462 6, L-Threonyl-L-phenylalanine-p-nitrobenzyl ester hydro-bromide, 34B, 274... 

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