Purification and some properties of beta-N-acetyl-D-glucosaminidase from the cabbage butterfly (Pieris rapae).
A beta-N-acetyl-D-glucosaminidase (NAGase) from the cabbage butterfly (Pieris rapae) was purified. The purified enzyme was a single band on polyacrylamide gel electrophoresis and the specific activity was determined to be 8715 U/mg. The molecular weight of whole enzyme was determined to be 106 kDa by gel filtration, and the result of SDS-PAGE showed that the enzyme was a heterodimer, which contained two subunits with different mass of 59.5 and 57.2 kDa. The optimum pH and optimum temperature of the enzyme for the hydrolysis of p-nitrophenyl-N-acetyl-beta-D-glucosaminide (pNP-NAG) were investigated to be at pH 6.2 and at 42 degrees C, respectively, and the Michaelis-Menten constant (K(m)) was determined to be 0.285 mM at pH 6.2 and 37 degrees C. The stability of the enzyme was investigated and the results showed that the enzyme was stable at the pH range from 4.0 to 9.0 and at the temperature below 45 degrees C. The activation energy was 83.86 kJ/mol. The reaction of this enzyme with pNP-NAG was judged to be Ordered Bi-Bi mechanism according to the inhibitory behaviors of the products. The ionization constant, pK(e), of ionizing group at the active site of the enzyme was found to be 5.20 at 39.0 degrees C, and the standard dissociation enthalpy (DeltaH(o)) was determined to be 2.18 kcal/mol. These results showed that the ionizing group of the enzyme active center was the carboxyl group. The results of chemical modification also suggested that carboxyl group was essential to the enzyme activity. Moreover, Zn(2+), Hg(2+), Cu(2+) had strongly inhibitory effects on the enzyme activity.
Shi
Yan
Y
Key Laboratory of the Ministry of Education for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University, Xiamen 361005, People's Republic of China.
Jiang
Zhe
Z
Han
Peng
P
Zheng
Guo-Xing
GX
Song
Kang-Kang
KK
Chen
Qing-Xi
QX
D000818:Animals; D001937:Brassica / Q000469:parasitology*; D002080:Butterflies / Q000201:enzymology*; D002412:Cations / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D013696:Temperature; D001619:beta-N-Acetylhexosaminidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
D002412:Cations; D008670:Metals; D001619:beta-N-Acetylhexosaminidases