ISHS Acta Horticulturae 650: I International Symposium on Saffron Biology and Biotechnology
DETECTION AND KINETIC PROPERTIES OF ALCOHOL DEHYDROGENASE IN DORMANT CORM OF CROCUS SATIVUS L. |
| Authors: | M. Hadizadeh, E. Keyhani |
| Keywords: | acetaldehyde, ethanol, kinetics, NAD+, NADH, substrate inhibition |
| Abstract:
An extract from dormant Crocus sativus L. corm was obtained which exhibited high alcohol dehydrogenase activity at optimum pH 8.5. The enzyme obeyed Michaelis-Menten kinetics with either ethanol or NAD+ as variable substrate and there appeared to be no cooperative interaction between the two active sites of this protein. For ethanol, Km was 13 ± 1 mM and Vmax was 6.2 ± 0.5 nmol.min-1 (mg protein)-1. For NAD+, Km was 1.12 ± 0.04 mM and Vmax was 8.2 ± 0.6 nmol.min-1 (mg protein)-1. Catalytic efficiency (calculated per mg protein in the extract) was (5 ± 0.5) x 10-4 min-1 with ethanol as the variable substrate, and it was (7.3 ± 0.6) x 10-3 min-1 with NAD+ as the variable substrate. When ethanol concentration was above 0.5 M, substrate inhibition was observed, while when NAD+ concentration was 0.005 M, substrate inhibition was also observed. Acetaldehyde inhibited ethanol oxidation rapidly; 50% inhibition was observed with 0.016 M acetaldehyde. C. sativus alcohol dehydrogenase did not use methanol, 2-propanol, nor isoamylalcohol as substrate, but used glycerol (2.8 ± 0.2 nmol.min-1 (mg protein)-1 and butanol (3.8 ± 0.3 nmol.min-1 (mg protein)-1). The enzyme being bifunctional, acetaldehyde and NADH could also be used as substrates. For acetaldehyde, Km was 1.9 ± 0.2 mM and Vmax was 30 ± 0.5 nmol.min-1 (mg protein)-1. For NADH, Km was 0.3 ± 0.03 mM and Vmax was 20 ± 1 nmol.min-1 (mg protein)-1. Catalytic efficiency (calculated per mg protein in the extract) was (1.6 ± 0.5) x 10-2 min-1 with acetaldehyde as the variable substrate, and it was (6.7 ± 1) x 10-2 min-1 with NADH as the variable substrate. | |
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