DETECTION AND KINETIC PROPERTIES OF ALCOHOL DEHYDROGENASE IN DORMANT CORM OF CROCUS SATIVUS L.

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, K_m was 13 ± 1 mM and V_max was 6.2 ± 0.5 nmol.min^-1 (mg protein)^-1. For NAD⁺, Km was 1.12 ± 0.04 mM and V_max 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, K_m was 1.9 ± 0.2 mM and V_max was 30 ± 0.5 nmol.min^-1 (mg protein)^-1. For NADH, Km was 0.3 ± 0.03 mM and V_max 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.

I International Symposium on Saffron Biology and Biotechnology

M. Hadizadeh, E. Keyhani

acetaldehyde, ethanol, kinetics, NAD⁺, NADH, substrate inhibition

https://doi.org/10.17660/ActaHortic.2004.650.13