TABLE 2

Kinetic parameters for PaDa-I and neutrally evolved UPO variantsa

Substrate and kinetic parameterPaDa-IVariant 16.5Variant 6.1Variant 7.1Variant 4.7
ABTS
    Km (mM)0.067 ± 0.0090.034 ± 0.0030.052 ± 0.030.09 ± 0.020.09 ± 0.01
    kcat (s−1)670 ± 37513 ± 12370 ± 7322 ± 43246 ± 15
    kcat/Km (mM−1 s−1)10,224 ± 1,02615,057 ± 2,0377,073 ± 2853,504 ± 7102,585 ± 244
DMP
    Km (mM)0.088 ± 0.0030.09 ± 0.010.29 ± 0.020.046 ± 0.0060.21 ± 0.01
    kcat (s−1)167 ± 276 ± 8162 ± 5108 ± 4264 ± 3
    kcat/Km (mM−1 s−1)1,899 ± 51777 ± 88543 ± 272,396 ± 2591,205 ± 25
NBD
    Km (mM)0.66 ± 0.211.77 ± 0.510.65 ± 0.20.20 ± 0.030.19 ± 0.07
    kcat (s−1)303 ± 40160 ± 26170 ± 20126 ± 4131 ± 8
    kcat/Km (mM−1 s−1)460 ± 10890 ± 11262 ± 50629 ± 77710 ± 222
Propranolol
    Km (mM)2.1 ± 0.12.5 ± 0.25.7 ± 2.12.1 ± 0.20.61 ± 0.09
    kcat (s−1)186 ± 625 ± 1255 ± 68167 ± 1081 ± 4
    kcat/Km (mM−1 s−1)90 ± 310.0 ± 0.544 ± 578 ± 4131 ± 13
Naphthalene
    Km (mM)0.38 ± 0.090.49 ± 0.090.59 ± 0.070.19 ± 0.050.48 ± 0.05
    kcat (s−1)162 ± 14119 ± 989 ± 497 ± 7127 ± 5
    kcat/Km (mM−1 s−1)421 ± 69243 ± 31150 ± 10520 ± 116264 ± 18
Veratryl alcohol
    Km (mM)12 ± 0.810 ± 19 ± 37 ± 120 ± 3
    kcat (s−1)256 ± 8141 ± 656 ± 7107 ± 5220 ± 22
    kcat/Km (mM−1 s−1)21 ± 113 ± 0.86 ± 116 ± 211 ± 1
Benzyl alcohol
    Km (mM)2.3 ± 0.32.5 ± 0.111 ± 22.3 ± 0.34.5 ± 0.3
    kcat (s−1)630 ± 26506 ± 9426 ± 50282 ± 13558 ± 17
    kcat/Km (mM−1 s−1)271 ± 26204 ± 839 ± 5121 ± 13124 ± 7
  • a For each substrate, reactions were performed in triplicate, with monitoring of the increases in absorption for ABTS (ε418 = 36,000 M−1 cm−1), NBD (ε425 = 9,700 M−1 cm−1), DMP (ε469 = 27,500 M−1 cm−1), propranolol (ε325 = 1,996 M−1 cm−1), naphthalene (ε303 = 2,010 M−1 cm−1), benzyl alcohol (ε280 = 1,400 M−1 cm−1), and veratryl alcohol (ε310 = 9,300 M−1 cm−1). Further details are presented in Materials and Methods.