Table 1 Kinetic parameters of MBP-fused hST3Gal1 and variants.

From: Structural and functional role of disulphide bonds and substrate binding residues of the human beta-galactoside alpha-2,3-sialyltransferase 1 (hST3Gal1)

 

Donor CMP-Neu5Ac

Acceptor Gal-β-1,3-GalNAc-α-O-Bn

KM (µm)

kcat (min−1)

kcat/KM (mm−1 min−1)

KM (µm)

kcat (min−1)

kcat/KM (mm−1 min−1)

ST3Gal1a

106 (±7)

201 (±5)

1896

26 (±1)

189 (±2)

7269

N147S (150)

668 (±159)b

75 (±9)

112

211 (±25)

48 (±2)

227

S148A (151)

481(±55)b

219 (±11)

455

105 (±12)

139 (±5)

1324

N170A (173)

184 (±29)

37 (±2)

201

96 (±16)

39 (±2)

406

Y191A (Y194)

876 (±206)b

38 (±5)

43

444 (±88)b

28 (±2)

63

Y230A (233)

205 (±18)

150 (±4)

732

854 (±105)b

178 (±11)

208

Y230F (233)

176 (±19)

162 (±6)

920

352 (±26)

208 (±5)

591

V315A (318)

180 (±23)

63 (±2)

350

1388 (±254)b

145 (±17)

105

V315Y (318)

271 (±51)

8 (±1)

28

549 (±78)b

12 (±1)

22

C59S/C64S

(62–67)

121 (±13)

135 (±4)

1116

20 (±2)

137 (±3)

6850

  1. Numbering corresponding to pST3Gal1 is given in parenthesis. Mean values and standard deviations of at least two replicates are shown.
  2. aValues for wild-type MBP-ST3Gal1 were taken from28. bFor these variants saturation was not observed at the highest donor and acceptor concentrations employed (1.2 and 1.4 mm, respectively) (Supplementary Information, Figs. S5 and S6).
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