1. [Elastase]
T Hayakawa, S Naruse, M Kitagawa, T Kondo Nihon Rinsho. 1995 May;53(5):1192-7.
Elastases are unique among the proteases in that they are capable of hydrolyzing the scleroprotein elastin. The enzymes include pancreatic elastases 1 (Protease E) and 2, and neutrophil elastase. These three elastases also have esterase and amidase activity toward synthetic substrates such as succinyl-trialanine-p-nitroanilide. Although the three enzymes are similar to each other in enzyme activity, they are quite different in immunoactivity. Therefore, each elastase has its own specific immunoassay either by RIA or EIA. Serum immunoreactive pancreatic elastases reflect disease conditions of pancreatic diseases, especially acute pancreatitis and pancreatic cancer. On the other hand, serum neutrophil elastase increases in various inflammatory diseases or conditions.
2. Kinetics of hydrolysis of Na-benzoyl-p-guanidino-L-phenylalanine p-nitroanilide by trypsin
H Tsunematsu, T Imamura, S Makisumi J Biochem. 1983 Jul;94(1):123-8. doi: 10.1093/oxfordjournals.jbchem.a134320.
A new chromogenic substrate, Na-benzoyl-p-guanidino-L-phenylalanine p-nitroanilide (Bz-GPA-pNA), was synthesized. This compound is a good substrate for bovine trypsin (Km = 1.56 X 10(-5) M, kcat = 0.081 s -1, at pH 8.2) and was hydrolyzed as fast as Na-benzoyl-L-arginine p-nitroanilide (Bz-Arg-pNA) with much the same kcat/Km values. But the values are two orders of magnitude smaller than those for ester substrates, Na-benzoyl-p-guanidino-L-phenylalanine ethyl ester (Bz-GPA-OEt) and Na-benzoyl-L-arginine ethyl ester (Bz-Arg-OEt). Substrate activation behavior was observed on tryptic hydrolysis of this new substrate in a substrate concentration range higher than about 5.0 X 10(-4) M in analogy with the trypsin-Bz-Arg-pNA system.
3. Effect of crowding by dextrans on the hydrolysis of N-Succinyl-L-phenyl-Ala-p-nitroanilide catalyzed by α-chymotrypsin
Isabel Pastor, Eudald Vilaseca, Sergio Madurga, Josep Lluís Garcés, Marta Cascante, Francesc Mas J Phys Chem B. 2011 Feb 10;115(5):1115-21. doi: 10.1021/jp105296c. Epub 2010 Dec 29.
Traditionally, studies on the diffusion-controlled reaction of biological macromolecules have been carried out in dilute solutions (in vitro). However, in an intracellular environment (in vivo), there is a high concentration of macromolecules, which results in nonspecific interactions (macromolecular crowding). This affects the kinetics and thermodynamics of the reactions that occur in these systems. In this paper, we study the crowding effect of large macromolecules on the reaction rates of the hydrolysis of N-succinyl-L-phenyl-Ala-p-nitroanilide catalyzed by α-chymotrypsin, by adding dextrans of various molecular weights to the reaction solutions. The results indicate that the volume occupied by the crowding agent, but not its size, plays an important role in the rate of this reaction. A v(max) decay and a K(m) increase were obtained when the dextran concentration in the sample was increased. The increase in K(m) can be attributed to the slowing of protein diffusion, due to the presence of crowding. Whereas the decrease in v(max) could be explained by the effect of mixed inhibition by product, which is enhanced in crowded media. As far as we know, this is the first reported experiment on the crowding effect in an enzymatic reaction with a mixed inhibition by product.
