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Table 2 Steady-state kinetic parameters of wild-type and mutant So BADH enzymes in the oxidation of APAL

From: Exploring the evolutionary route of the acquisition of betaine aldehyde dehydrogenase activity by plant ALDH10 enzymes: implications for the synthesis of the osmoprotectant glycine betaine

  Kinetic parameters
Enzyme Variable substrate k cat (s-1) Km(μM) k cat /Km(mM-1s-1)
  APAL    
Wild type   0.99 ± 0.04 3.9 ± 0.1 256 ± 5
A441C   0.67 ± 0.02 0.72 ± 0.16 931 ± 188
A441S   1.12 ± 0.00 2.0 ± 0.5 550 ± 142
A441T   1.50 ± 0.12 4.6 ± 0.5 326 ± 13
A441V   0.52 ± 0.10 1.1 ± 0.3 473 ± 216
A441F   0.34 ± 0.04 3.7 ± 0.0 92 ± 11
A441I   1.85 ± 0.06 4.8 ± 1.3 375 ± 80
  NAD +    
Wild type   0.99 ± 0.04 4.0 ± 0.1 250 ± 7
A441C   0.89 ± 0.00 2.6 ± 0.5 342 ± 76
A441S   0.88 ± 0.02 2.8 ± 0.0 314 ± 13
A441T   0.76 ± 0.02 4.0 ± 0.4 190 ± 16
A441V   0.54 ± 0.06 1.7 ± 0.2 318 ± 2
A441F   0.43 ± 0.01 5.9 ± 1.4 73 ± 17
A441I   2.11 ± 0.01 5.5 ± 0.1 382 ± 9
  1. Initial velocities were obtained at 30°C in 50 mM HEPES-KOH buffer, pH 8.0, containing 0.1 mM EDTA. In the experiments with variable APAL, the fixed concentration of NAD+ was 0.2 mM, and in the experiments with variable NAD+ the fixed APAL concentrations were at least 10-times their appKm values estimated for each enzyme at fixed 0.2 mM NAD+. The apparent kinetic parameters were estimated by non-linear regression fit of the experimental data to the Michaelis-Menten equation (saturation by NAD+ at fixed APAL) or to Equation 1 given in the main text (saturation by APAL at fixed NAD+). The values given in the Table are the mean ± standard deviation of the kinetic parameters estimated in two duplicate saturation experiments performed with enzymes from two different purification batches. Values for k cat are expressed per enzyme subunit. Substrate inhibition constants for APAL are not given because they could not be accurately estimated in the concentration range used in these experiments, but the observed degree of inhibition by high APAL concentrations was roughly the same in all the enzymes.