Internal Truncation Mutants of a Family 3 ꢀ-Glucosidase
1117
Discussion
sequence analysis predicted that most of the region
(Leu426–Glu446) formed an ꢁ-helix structure as shown
in Fig. 3. Thus, it is suggested that the ꢁ-helix plays an
important role in maintaining a strong affinity toward
hydrophobic compounds at the acceptor site.
The length of the non-homologous region of Family 3
ꢀ-glucosidase varies with the enzyme. But the effect of
length on enzymatic characteristics is still unclear. Cbg1
possesses a longer non-homologous region than other
enzymes. To investigate the role of the long non-
homologous region located at the center of Cbg1, five
truncated mutants were constructed and expressed. The
non-homologous region was considered to be located
between the N-terminal ðꢁ=ꢀÞ8 TIM barrel domain and
the C-terminal six-stranded ꢀ sandwich, with the
catalytic center being at their interface.4,5,7) All the
truncated mutants prepared in this study were catalyti-
cally active, indicating that the non-homologous region
is not essential for the expression of ꢀ-glucosidase.
However, truncation resulted in shifts in optimum pH,
decreases in thermal stability, and decreases in trans-
glycosyl activity.
The truncated mutants showed lower optimum pH
than did Cbg1. The pH-activity curves narrowed with
truncation (Fig. 5). The narrow curve was most pro-
nounced in the case of Cbg-d62. The widths at the mid-
points of the curves were approximately 4.5 (Cbg1), 4.2
(Cbg1-d4), 3.2 (Cbg1-d31), 1.5 (Cbg1-d62), 2.5 (Cbg1-
d89), and 3.3 (Cbg1-d119). These curves are narrow
only on the basic side. There was no significant
expansion on the acidic side. The acidic and basic sides
of the pH-activity curve are regulated by dissociation/
association of the nucleophile residue and the proton
donor residue respectively. This explains the shift in
optimum pH with decreases in the pKa of the proton
donor Glu616.
When the truncation was long, the thermal stability
and pH optimum partially recovered. These results
strongly suggest that the changes in the properties were
due to structural perturbation caused by the truncation,
rather than by a direct role of the non-homologous
region.
Cbg1 exhibits a very low Km value toward pNP-Glc.
The high transglycosyl activity on hydrophobic alcohols
suggests that the enzyme has a strong affinity toward
hydrophobic compounds at the acceptor site. The
truncation caused an increase in the Km values of
pNP-Glc and also a deficiency in transglycosylation.
The transglycosylation was not recovered by the longer
truncation, suggesting that this is the role of the non-
homologous region.
Acknowledgments
We are grateful to the United Nations University and
Kirin Brewery Company for granting a UNU–Kirin
Fellowship to LY.
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