N-linked glycosylation influences on the catalytic and biochemical properties of Penicillium purpurogenum β-d-glucuronidase.
To study the influence of N-linked carbohydrate moiety on the catalytic and biochemical properties of glycosylated enzyme, a recombinant β-d-glucuronidase (PGUS-P) from Penicillium purpurogenum as a model glycoprotein, was deglycosylated with peptide-N-glycosidase F (PNGase-F) under native conditions. The enzymatic deglycosylation procedure resulted in the complete removal of carbohydrate moiety. Compared with the glycosylated PGUS-P, the deglycosylated PGUS-P exhibited 20-70% higher activity (p<0.05) within pH 6-9, but 15-45% lower activity (p<0.05) at 45-70°C. The apparent decrease in the thermal stability of the deglycosylated enzyme was reflected by a decrease in the denaturation temperature (T(d)) values determined by differential scanning calorimetry (DSC). The removal of N-linked glycans also reduced enzyme's sensitivity to certain metal ions. The deglycosylated PGUS-P displayed lower K(m) vaules, but higher k(cat)/K(m) ratios than the glycosylated isoform towards glycyrrhizin. The consequent conformational changes were also determined by circular dichroism (CD) and fluorescence spectroscopy which revealed no significant difference in the secondary but a slight dissimilarity between the tertiary structures of both isoforms of PGUS-P.
Institute of Bioengineering, Zhejiang University of Technology, Hangzhou 310014, People's Republic of China.
Shuping
S
Zou
Luping
L
Xie
Yanli
Y
Liu
Imdad
I
Kaleem
Guifeng
G
Zhang
Chun
C
Li
D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D005966:Glucuronidase / Q000302:isolation & purification / Q000378:metabolism*; D006031:Glycosylation; D019695:Glycyrrhizic Acid / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D010407:Penicillium / Q000201:enzymology*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature
D019695:Glycyrrhizic Acid; D005966:Glucuronidase