Table 1 Apparent kinetic parameters for Cps2L substrates with dTTP
Parameter
127
4
5
6
b-L-Araf-1-P27
Vmax (mM minꢁ1
Km (mM)
)
2.818
0.4321
124
0.198
0.00166
0.493
176
0.00849
0.0000483
0.1022
107
0.00335
0.0000313
0.2801
90
0.00218
0.0000242
139
27.6
0.199
kcat (minꢁ1
)
kcat/Km (mMꢁ1 minꢁ1
)
to the physiological substrate.33 In contrast, the phosphonate
analogue 4, exhibited a 120-fold decrease in kcat/Km. The
presence of the methylene functionality as a non-scissile
isosteric analogue of the glycosidic linkage in these sugar
nucleotide analogues (7–9), and the facile access to these
compounds by enzymatic coupling, will enable their use as
substrates for subsequent sugar nucleotide processing enzymes
to generate specific isosteric glycosyltransferase inhibitors.
In conclusion, nucleotidylyltransferases have been shown to
solve the issues of low yields and sluggish reaction times for
the formation of phosphono analogues of sugar nucleotides.
Cps2L is the first nucleotidylyltransferase to couple dTTP and
UTP nucleotides with a phosphonate analogue of glucose-
1-phosphate to produce phosphono analogues of dTDP- and
UDP-sugars. It is also capable of producing a phosphono
analogue of dTDP-Gal. The enzymatic production of these
products indicate that increases in the second ionization
constant (pKa2) does not significantly hinder the catalytic
activity. In the case of the galacto-configured substrates, the
phosphonate is a better substrate than the phosphate. Further-
more, the ready conversion of the phosphono analogue
of glucose-1-phosphate to phosphono sugar nucleotide
analogues indicates the potential of efficient enzymatic
transformations to generate novel glycosyltransferase probes.
We thank the Canadian Institutes of Health Research, the
Natural Sciences and Engineering Research Council, The
Royal Society (UK) and the Mizutani Glycoscience Founda-
tion of Japan for funding. We thank Jessica Pearson for
purification of the enzyme.
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240 | Chem. Commun., 2009, 238–240