UNG T(°C) Vmax(nmol/min/mg) kcat(min-1) KM(μM) kcat/KM(min-1μM-1)
cUNG[35] 37 49135 ± 1626 1241 0.8 ± 0.09 1499
22 24439 ± 555 617 0.8 ± 0.06 795
15 11064 ± 374 279 0.5 ± 0.07 607
A266T 37 53467 ± 1079 1352 0.6 ± 0.04 2282
22 24994 ± 464 632 1.0 ± 0.05 641
15 10006 ± 170 253 0.7 ± 0.04 389
V267A 37 47476 ± 1363 1197 0.5 ± 0.06 2507
22 15218 ± 313 384 0.6 ± 0.04 685
15 5867 ± 113 148 0.3 ± 0.03 500
Double (A266T/V267A) 37 68898 ± 1455 1740 0.7 ± 0.05 2500
22 20891 ± 320 528 0.8 ± 0.04 686
15 10805 ± 249 273 0.5 ± 0.05 549
A274V 37 37379 ± 611 944 1.1 ± 0.05 830
22 11746 ± 244 297 0.8 ± 0.06 381
15 8991 ± 286 227 1.3 ± 0.11 174
Triple1
(A266T/V267A/A274V)		 37 61843 ± 2879 1562 0.7 ± 0.12 2199
22 30544 ± 733 771 0.8 ± 0.07 929
15 14550 ± 509 367 0.7 ± 0.09 496
H275Y [35] 37 23590 ± 859 596 0.6 ± 0.09 1017
22 11974 ± 553 302 0.6 ± 0.11 509
15 6166 ± 384 156 0.7 ± 0.16 224
Leuloop
(A266T/V267A/A274V/H275Y)		 37 26603 ± 1293 672 0.7 ± 0.12 1018
22 10415 ± 282 263 0.5 ± 0.06 548
15 6007 ± 273 152 0.5 ± 0.11 292
L279F 37 31674 ± 321 800 0.5 ± 0.02 1459
22 11944 ± 145 302 0.5 ± 0.03 611
15 7977 ± 252 201 0.6 ± 0.08 342
Triple2
(A274V/H275Y/L279F)		 37 30569 ± 676 713 0.8 ± 0.06 919
22 7551 ± 160 176 0.8 ± 0.05 215
15 4352 ± 79 102 0.7 ± 0.04 146
hUNG[35] 37 25347 ± 1256 647 2.1 ± 0.23 309
22 12530 ± 740 320 2.4 ± 0.29 135
15 5580 ± 399 142 2.2 ± 0.34 66
Table 2: Kinetic constants of the interactions of cUNG and mutants with substitutions in the Leucine-loop with uracil (3H+ labelled) incorporated calf thymus DNA. The assay was performed at 15, 22 and 37°C in 25 mM Tris/HCl (pH 7.5 at 25°C), 50 mM NaCl, 1 mM EDTA. The Vmax and Km were extracted by fitting the velocity data obtained at 8 different substrate concentrations on the Michaelis-Menten equation using the enzyme kinetics module in Sigma Plot. The standard error (SD) for the fit is indicated.