P-Nitrophenyl galactosides

Figure 4.8 Optical density variation with the concentration of [p-nitrophenyl-/ -galactoside]. The optical densities correspond to 2 ml of hydrolysis solution.

Typical effects of temperature on catalysis will be illustrated with the reaction of / -galactosidase and o- and p-nitrophenyl galactosides (18). The minimum reaction pathway may be represented by Scheme 1 in which ES is the noncovalent Michaelis complex, and EG represents an enzyme-galactose intermediate (25,26). 

Hydrolysis of p-Nitrophenyl-/ -D-Galactoside with / -Galactosidase from E coli... 

The purpose of the present experiments is to characterize kinetics parameters of p-nitrophenyl-/3-D-galactoside hydrolysis with / -galactosidase. Students will find out how to use absorption spectroscopy to study enzymatic properties of an enzyme (see also Murata et al. 2003). Before entering the lab, students should be able to explain the Beer-Lambert-Bouguer law and the basics of enzymology. 

Kinetics of p-nitrophenyl-fi-D-galactoside hydrolysis with (3 -galactosidase... 

First, we can see that the measured ODs fit within the linear part of the plot of Figure 4.4. The ODs correspond to PNP resulting from p-nitrophenyl-/3-D-galactoside hydrolysis. From the slope of the linear part of the kinetics, we can calculate the hydrolysis velocity, i.e., the PNP concentration formed per minute. 

Figure 4.9 Double reciprocal plot of PNP optical density vs. [p-nitrophenyl-jS-D-galactoside] at high...

D. F. Day and W. Yaphe, Enzymatic hydrolysis of agar Purification and characterization of neoagar-obiose hydrolase and p-nitrophenyl a-galactoside hydrolase, Can. J. Microbiol., 21 (1975) 1512-1518. 

Replacement of the D-galactosidic, anomeric oxygen atom by a sulfur atom was reported to lead to complete loss of activity, although the affinity for the enzyme remains unaltered."A re-investigation showed that 0- and p-nitrophenyl l-thio-/3-D-galactopyranosides were split at a high concentration of enzyme. At 20° and pH 7.6, in 2-amino-2-(hydroxy-... 

Fig. 8.—Activation of Hydrolysis of o-Nitrophenyl /3-D-GaIactoside by Sodium Chloride. (0.05 M 2-amino-2-(hydroxymethyl)-l,3-propanediol-HCl buffer, pH 7.6 20° 3.33 X 10 M o-nitrophenyl p-n-galactoside. M = concentration of NaCl in test Mo = optimum NaCl concentration (3.2 X 10 (If) AE = increase in optical density (405 mp) in 2 minutes.)...

Sodium ions are better activators for the hydrolysis of o-nitrophenyl /S-D-galactoside and o-nitrophenyl a-L-arabinoside, and K ions for that of lactose and p-nitrophenyl /3-o-galactoside (compare Ref. 20). More substrates and alkali ions will have to be studied before any correlation or generalization can be made. 

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