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Table 2 Kinetic parameters of PirXI variants

From: Structure-based directed evolution improves S. cerevisiae growth on xylose by influencing in vivo enzyme performance

PirXI variantsMetalKact (µM)cKinetic parametersa
kcat (s−1)KM (mM)kcat/KM (s−1 M−1)
WTMg2+130 ± 72.2 ± 0.17.1 ± 0.5310
Mn2+0.44 ± 0.056.9 ± 0.26.2 ± 0.71110
Ca2+230 ± 200.8 ± 0.11360 ± 1600.4b
V270A–A273GMg2+160 ± 141.8 ± 0.111.6 ± 1.6155
Mn2+0.39 ± 0.052.8 ± 0.19 ± 1310
Ca2+140 ± 120.28 ± 0.011760 ± 1200.18b
S141N–T142S–A143S–G174AMg2+126 ± 101.6 ± 0.227 ± 960
Mn2+2.4 ± 0.54.9 ± 137 ± 1.5132
Ca2+96.5 ± 3.50.06 ± 0.011200 ± 3000.03b
E15D–T142SMg2+190 ± 101.9 ± 0.122 ± 286
Mn2+2.1 ± 0.35 ± 0.253 ± 494
Ca2+50 ± 10> 0.02N.A.0.018b
N338CMg2+112 ± 74.2 ± 0.222 ± 2191
Mn2+1.3 ± 0.37.2 ± 0.229 ± 1.5248
Ca2+163 ± 6.50.78 ± 0.22800 ± 8000.2b
  1. aAll assays were performed at 30 °C in 20 mM MOPS buffer, pH 7. Values are averages from three (wild type, V270A–A273G PirXI) or two (S141N–T142S–A143S–G174A, E15D–T142S and N338C PirXI) independent measurements. The margins represent standard deviations
  2. bCatalytic efficiencies obtained from slopes of Michaelis–Menten plots at [S] KM because of the high KM for xylose in the presence of Ca2+ as activating metal
  3. cThe Kact value is defined as the metal concentration giving half-maximal activity, as measured with a saturating concentration of xylose (100 mM in case of Mg+ and Mn2+, 400 mM in case of Ca2+)